Western Michigan University ScholarWorks at WMU
Master's Theses Graduate College
8-2008
Optical and Electrochemical Molecular and Nanoscale Sensors for the Selective Detection of Organophosphorus Based Pesticides
Chandrima De
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Recommended Citation De, Chandrima, "Optical and Electrochemical Molecular and Nanoscale Sensors for the Selective Detection of Organophosphorus Based Pesticides" (2008). Master's Theses. 4545. https://scholarworks.wmich.edu/masters_theses/4545
This Masters Thesis-Open Access is brought to you for free and open access by the Graduate College at ScholarWorks at WMU. It has been accepted for inclusion in Master's Theses by an authorized administrator of ScholarWorks at WMU. For more information, please contact [email protected]. OPTICAL AND ELECTROCHEMICAL MOLECULAR AND NANOSCALE SENSORS FOR THE SELECTIVE DETECTION OF ORGANOPHOSPHORUS BASED PESTICIDES
by
Chandrima De
A Thesis Submitted to the Faculty of The Graduate College in partial fulfillment of the requirements forthe Degree of Master of Science Department of Chemistry
WesternMichigan University Kalamazoo, Michigan August 2008 © 2008 Chandrima De ACKNOWLEDGEMENTS
I would like to take this opportunity to express my gratitude to individuals for
their help, support and motivation for the successfulcompletion of this research.
Honestly, I do not have words to express my gratitude to my research advisor, mentor, and chair of my committee, Dr. Sherine Obare for all her guidance, support, expertise, and encouragement during my entire stay in Western Michigan University.
She has been the constant source of inspiration throughout my project work. Her commitment to education and research, in addition to her emphasis on strong personality development are deeply appreciated.
I would also like to thank my other research committee members, Dr. Herb
Fynewever, and Dr. Yirong Mo for their significant contribution in my graduate research, and invaluable advices towards the completion of this project. I am highly grateful to all the members of the Obare research group at Western Michigan
University (WMU) and University of North Carolina at Charlotte (UNCC) for their enjoyable company, constant support and encouragement. Specifically, I would like to thank Christopher Ciptadjaya, Ruel Freemantle, and Wen Guo for their help with my experiments and making my stay in graduate school as a memorable experience.
In addition, I am grateful to Dr. Sherine Obare for the financial support during my graduate studies.
11 Acknowledgements-Continued
I am thankful to all the faculty, graduate students, and staffof the Department of chemistry at Western Michigan University for their help, encouragement, and constant support during my entire graduate work. The Department of Chemistry is highly acknowledged for giving me an opportunity to continue my higher education and financial assistance to fulfill my childhood dreams.
Last but not the least; I would like to express my gratefulness to my entire family for always supporting me and believing in me. My mother, Mrs. Debjani Sen, father, Mr. Sabyasachi Sen, sister, Miss Debleena Sen, and grandmother, Mrs.
Provaboti Sen has been a source of inspiration, encouragement, and motivation in my life. I would like to convey heartfelt thanks to my husband, Dr. Sinjan De, mother-in law, Mrs. Lakhshmi De, and father-in-law, Mr. Ajit Kumar De for their understanding, and encouragement to pursue my graduate degree. I am highly grateful to all my family members for their patience, eternal support, and above all believing in me. Without their support in every aspect of my life, this work would not have been possible.
Chandrima De
111 OPTICAL AND ELECTROCHEMICAL MOLECULAR AND NANOSCALE SENSORS FOR THE SELECTIVE DETECTION OF ORGANOPHOSPHORUS BASED PESTICIDES
Chandrima De, M.S..
WesternMichigan University, 2008
Organophosphorus based pesticides are highly toxic and represent senous environmental concerns. These compounds tend to easily enter ground water and cause drastic pollution. The toxicity levels of the organophosphorus pesticides however are based on their structures, and hence methods for their selective detection are in high demand. The creation of new materials for sensing and actuation requires careful manipulation of the responsive units required to control analyte selectivity.
We have successfully designed and synthesized a molecular sensor based on the family of stilbenes that not only can detect organophosphorus pesticides, but also distinguish between them. Four different pesticides have been investigated, namely, ethion, malathion, fenthion, and parathion. The sensors show significant differences in the optical output and electrochemical signal by interacting with the organophosphorus pesticides. The dual signal transduction is advantageous because it minimizes false positives. Apart from molecular sensors, we have also developed a series of gold nanoparticle-based sensors for detection of organophosphorus pesticides. Both the molecular and nanomaterials as sensors operate in real time and have parts-per-million detection limits. TABLE OF CONTENTS
ACKNOWLEDGEMENTS...... 11
LIST OF TABLES...... :...... Vlll
LIST OF FIGURES...... ···•···································· ··································•····••··· lX
LIST OF SCHEMES...... XlV
CHAPTER
1. INTRODUCTION TO ORGANOPHOSPHORUS COJ\1POUNDS AND THE NEED FOR NOVEL SENSORS FOR THEIR DETECTION...... 1
1.1 Sensors forDetection of Organic-Based Environmental Contaminants ...... 1
1.2 Organophosphorus (OP) Pesticides...... 3
1.2.1 Organophosphorus Pesticides and the Environment ..... 3
1.2.2 Structures of Organophosphorus Compounds...... 6
1.2.3 Health Hazards fromOrganophosphorus Pesticides ..... 8
1.2.4 Mechanism oflnhibition of Acetylcholinesterase...... 10
1. 3 Advances in Detection of Organophosphorus Pesticides...... 14
1. 3. 1 Enzyme Based Detection Methods...... 15
1. 3. 2 Chromatographic Detection Methods...... 18
1.4 The Need...... 20
1.5 Our Project...... 21
1V Table ofContents-Continued
CHAPTER
1.5.1 Selective Colorimetric and Electrochemical Detection ofOrganophosphorus Based Pesticides...... 24
1.5.2 Application ofNanoscale Materials as Sensors toward Detection ofOrganophosphorus Based Pesticides ...... 29
1.6 Research Objectives and Hypothesis...... 33
1. 7 References...... 35
2. SELECTIVE COLORIMETRIC AND ELECTROCHEMICAL DETECTION OF ORGANOPHOSPHORUS BASED PESTICIDES...... 43
2.1 Introduction...... 43
2.1.1 Chemical Sensors and Molecular Recognition...... 43
2.1.2 Advances in Optical Methods of Detection of Organophosphorus Compounds...... 45
2.2 Experimental Section...... 47
2.2.1 Materials andInstrumentation ...... 47
2.2.2 Synthesis ofDQA ...... 48
2.2.3 UV-visible Absorbance Measurements ...... 49
2.2.4 Fluorescence Measurements...... 50
2.2.5 Electrochemical Measurements...... 50
2.2.6 Computational Modeling...... 51
2.2.7 I HAND 31 PNMR Measurements ...... 51
V Table of Contents-Continued
CHAPTER
2.3 Results and Discussion ...... 52
2.3.1 UV-visible Absorbance Spectroscopy Studies...... 54
2.3.2 Fluorescence Spectroscopy...... 60
2.3.3 Electrochemical Measurements ...... 65
2.3.4 NMR Studies...... 68
2.4 Investigation on the Binding Sites in DQA ...... 71
2.5 Mechanism oflnteraction ...... 75
2.6 Conclusions...... 78
2.7 References...... 79
3. WAVELENGTH-SPECIFIC SURFACE PLASMON RESONANCE SHIFTS IN GOLD NANOPARTICLES CAUSED BY ORGANOPHOSPHORUS PESTICIDES...... 83
3.1 Introduction...... 83
3.1.1 Advantages of using Gold Nanoparticles as Sensors toward Detection of Organophosphorus Pesticides ...... 83
3.2 Experimental Section...... 88
3.2.1 Materials ...... 88
3.2.2 Transmission Electron Microscopy (TEM) ...... 88
3.2.3 UV-visible AbsorptionSpectroscopy ...... 88
Vl Table of Contents-Continued
CHAPTER
3.2.4 Synthesis of 4 nm Au Nanoparticles...... 89
3.2.5 Synthesis of 13 and 30 nm Au Nanoparticles...... 90
3.3 Results and Discussion ...... 92
3.3.1 Interaction of the 4 nm Gold Nanoparticles with Ethion, Malathion, and Fenthion ...... 92
3.3.2 Interaction of the 13 nm Gold Nanoparticles with Ethion, Malathion, and Fenthion ...... 98
3.3.3 Interaction of the 30 nm Gold Nanoparticles with Ethion, Malathion, and Fenthion ...... 104
3.4 Sum1nary...... 108
3.5 Conclusion ...... 111
3.6 References...... 113
BIBLIOGRAPHY...... 117
Vll LIST OF TABLES
I. List of the 48 widely applied organophosphorus pesticides...... 3
2. List of the most toxic OP pesticides with their mammalian toxicities ...... I 0
3. Physicochemical Properties of the Selected Organophosphorus Pesticides...... 22
4. Summary of results for the interaction of DQA with the OP pesticides...... 59
5. Summary of results for the interaction of the Au NPs of three different diameters with the OP pesticides ...... 108
VIII LIST OF FIGURES
1. Schematic representation of the possible routes of environmental exposure of organophosphurus pesticides to humans and wildlife ...... 6
2. Examples of structures of main classes of OP compounds...... 7
3. Structures of common organophosphorL1S pesticides...... 8
4. Structure of HETP...... 9
5. (A) Inhibition scheme of acetylcholinesterase (AChE) by organophosphates (B) Mechanism of inhibition of acetylcholinesterase by OP pesticides...... 11
6. Structures of some Nerve Gases...... 13
7. (A) Structure of fluoresceinisothiocyanate (FITC) at different pH, (B) its relative fluorescence intensity at selected pH values...... 16
8. Mechanism for the hydrolysis of paraoxon by OPH ...... 17
9. Structures of the organophosphorus pesticides investigated for the project ...... 21
I 0. Mechanism of the chemically reactive sensor developed by Pilato et al...... 26
11. The mechanistic scheme of the chemosensor developed by Swager et al ...... 27
12. The chemically reactive sensor reported by Rebek et. al...... 28
13. Coumarin l, the fluorescent compound and competitive inhibitor as reported by Sin1onian et. al ...... 28
IX List of Figures-Continued
14. (A) Schematic of the anion-sensing by Au NP functionalized with zinc porphyrin. (B) Scheme of the interaction of MUA capped Au NP with heavy metal ions ...... 31
15. Schematic representation of the process of molecular recognition: a receptor is synthesized with recognition units or binding sites for the substrate...... 45
16. Structure of the molecular sensor ...... 4 7
17. UV-visible absorbance spectrum of DQA in acetonitrile ...... 53
18. Fluorescence spectrum of DQA in acetonitrile ...... 53
19. Changes in the UV-visible absorbance titration profile of DQA in the presence of increasing concentrations of ethion ...... 55
20. Changes in the UY-visible absorbance titration profile of DQA in the presence of increasing concentrations of malathion ...... 56
21. Changes in the UV-visible absorbance titration profile of DQA in the presence of increasing concentrations of fenthion...... 57
22. Changes in the UV-visible absorbance titration profile of DQA in the presence of increasing concentrations of parathion ...... 58
23. Changes in the fluorescence titration profile of DQA in the presence . . . . ,., o f mcreasmg concentrations o t~ et h1011 ...... :,: ...... •.... 61
24. Changes in the fluorescence titration profile of OQA in the presence of increasing concentrations of malathion ...... :...... 62
25. Changes in the fluorescence titration profileof DQA in the presence of increasing concentrations of fenthion ...... :..... 63
26. Changes in the fluorescence titration profileof DQA in the presence of increasing concentrations of parathion ...... 64
X List of Figures-Continued
27. Cyclic voltammogram of 8.75x I 0-4 M DQA alone in acetonitrile ...... 66
28. Changes in redox properties of DQA upon addition of 7x10- 4 M n1alathion...... 67
29. Changes in redox properties of DQA upon addition of 7x I0- 3 M parathion ...... 68
30. Structure of DQA showing the protons susceptible to undergo chemical shifts upon binding with the OP pesticides...... 69
31. 1 H NMR shifts in the quinaldine nitrogen of 3.3xJ0-5 M ofDQA in CO3CN upon addition of ethion in (a) 1:0.4 (b) I:0.8, and (c) I: I n1olar ratio ...... 70
32. Molecular electrostatic potential ofDQA calculated by Gaussian 03 softwarepackage...... 72
33. Chemical shifts in H I with gradual addition of ethion...... 73
34. (A) Structure of QPE, and (B) Changes in the UV-visible absorbance titration profileof QPE with the addition of ethion, inset showing the colors of the solutions before and aftertitration ...... 74
35. Structure of the leaving group in the case of parathion interaction ...... 77
36. Detection scheme for OP pesticides with gold nanoparticles capped with citrate ...... 86
3 7. Structures of the investigated Organophospborus Pesticides ...... 87
38. (A) TEM images of 4 nm gold nanoparticles (B) UV-visible absorbance spectrum for4 nm gold nanoparticles...... 90
39. (A) TEM images of 13 nm gold nanoparticles (B) UV-visible absorbance spectrum for 13 nm gold nanoparticles ...... 91
40. (A) TEM images of 30 11111 gold nanoparticles (B) UV-visible absorbance spectrum for30 11111 gold nanoparticles...... 91
XI List of Figures-Continued
41. UV-visible absorbance spectra of 4 nm Au nanoparticles in the presence of increasing concentrations of ethion ...... 93
42. UV-visible absorbance spectra of 4 nm Au nanoparticles in the presence of increasing concentrations of malathion ...... 94
43. UV-visible absorbance spectra of 4 nm Au nanoparticles in the presence of increasing concentrations of fenthion...... 96
44. TEM image of (A) 4 nm Au NPs in the absence of any OP pesticides, and (B), (C), and (D) after titration with ethion, malathion, and fenthion respectively, indicating particle aggregation ...... 97
45. Color changes observed when OP pesticides ethion, malathion, and fenthion were added to the 4 nm Au nanoparticles...... 98
46. Changes in the SPR peak of 13 nm Au nanoparticles titrated with ethion...... 100
47. Changes in the SPR peak of 13 nm Au nanoparticles with increase in malathion concentrations ...... 101
48. Changes in the SPR peak of 13 nm Au nanoparticles in the with increase in fenthion concentrations ...... ] 02
49. TEM image of (A) 13 nm Au NPs before, and (B), (C), and (D) after titration with ethion, malathion, and fenthionrespectively. Particles have aggregated after titration ...... 103
50. Color changes observed with 13 nm Au nps after addition of the corresponding OP pesticides ...... I 04
51. UV-visible absorbance spectra of 30 nm Au nanoparticles with increasing concentrations of ethion ...... 105
52. UV-visible absorbance spectra of 30 nm Au nanoparticles with increasing concentrations of malathion ...... 106
XII List of Figures-Continued
53. UV-visible absorbance spectra of 30 nm Au nanoparticles with increasing concentrations of fenthion ...... l 07
54. TEM images of the~ 30nm Au NPs before addition ofOP ...... 107
55. (A) SPR shifts in 4 nm Au NPs caused by ethion, malathion, and fenthion (B) SPR shifts in 13 nm Au NPs caused by ethion, malathion, and fenthion ...... 109
56. Structure of Paraoxon ...... 11 I
XIII LIST OF SCHEMES
I. Synthesis of Dimethyl-[4-(2-quinolin-2-yl-vinyl)-phenyl]-amine (DQA) ...... 48
2. Mechanism of interaction of DQA with the OP pesticides in general ...... 75
3. Mechanism of interaction of DQA with fenthion...... 77
XIV CHAPTER 1
INTRODUCTION TO ORGANOPHOSPHORUS COMPOUNDS AND THE NEED FOR NOVEL SENSORS FOR THEIR DETECTION
1.1 Sensors for Detection of Organic-Based Environmental Contaminants
Organic chemical contamination has become a worldwide problem. Modern industrialized societies have developed thousands of synthetic organic compounds and volatile organic compounds (e.g., benzene) for several uses. The most important ones include plastics, lubricants, refrigerants, fuels, solvents, preservatives, and pesticides. However, as a result of improper practices, these contaminants enter the environment and cause air, water, and soil pollution. For instance, pesticides and herbicides are applied directly to plants and soils, and incidental releases of other contaminants can originate from spills, leaking pipes, underground storage tanks, waste dumps, and waste repositories. Some of these contaminants have long half lives and thus remain in the environment. They migrate through large regions of soil until they reach water resources, where they may present an ecological or human health threat. In fact organisms including humans are affected by various chemicals through either inhalation or ingestion that have entered water bodies, volatilized in the air, been taken up by vegetation, or remain in the soil. These contaminants pose serious health hazards. The civilian, commercial, and defense sectors of most advanced industrialized nations as well as developing nations are facing tremendous
1 environmental concerns due to these various contaminants. Environmental monitoring 1s required to protect the public and the environment from toxic contaminants and pathogens that can be released into a variety of media including air, soil, and water. The United States Environmental Protection Agency (U.S. EPA) has imposed strict regulations on the concentrations of many environmental contaminants in air and water. 1 However, current monitoring methods are costly and time-intensive, and limitations in sampling and analytical techniques exist.2 Thus, there is a great demand for development of quick and simple methods for the detection of these compounds. Table 1 lists the names of the 48 most commonly used
3 organophosphorus pesticides. This thesis addresses a class of organic pollutants namely organophosphorus based pesticides. Our goal is to design portable sensors for organophosphorus based pesticides that can provide rapid responses (relative to current methods and technologies), ease of operation (for field use), and sufficient detection limits.
2 Compound Compound Azinphos-Me Diazoxon Carbophenoth ion Dichlorvos Dimethoate Dicrotophos Disulfoton Mevinphos Malathion Monocrotophos Phorate Naled Phosalone Phosphamidon Phosmet Tetrachlorvinphos Terbufos Demeton-S Thiometon Oxydemeton-Me Chlorpyrifos Acephate Chlorpyrifos-Me EPN Demeton-O Ethephon Diazinon Ethoprop Fenitrothion Fenamiphos Fensulfothion Fonofos Fenthion Fosamine Ammonium lsazofos Glyphosate Methyl Parathion Metamidphos Parathion Profenofos Pirimiphos-Me Sulprofos Pi rimiphos-Ethyl Tribufos Temephos Trichlorfon Tolelofos-Me Trichloronal
Table 1 List of the 48 widely applied organophosphorus pesticides.3
1.2 Organophosphorus (OP) Pesticides
1.2.1 Organophosphorus Pesticides and the Environment
Pesticides are used to describe any chemical that kills or slows down the
growth of an undesirable organism. Pesticides include herbicides, insecticides,
3 fungicides, and nematocides. In order to meet the nutritional needs of an increasing world population, there is no doubt about the necessity of application of various pesticides to crops and animals. It is believed nowadays that application of synthetic pesticides is the most effective method of controlling insects that affect crop growth.4
It has been estimated that approximately 40,000 new potentially poisonous products enter the market each year.5
Organophosphorus (OP) pesticides constitute the most widely used insecticides available today. This class of compounds has achieved enormous commercial success as a key component in the arsenal of agricultural pesticides and herbicides, and is currently an integral element of modem agriculture. According to the EPA, about 70% of the insecticides in current use in the US are organophosphorus pesticides. 1 They were developed to replace organohalide pesticides in the late 1950' s because of the fact that these OPs are relatively easier to degrade via microbial or environmental processes. Unlike organohalide pesticides, the OPs do not bioaccumulate due to their rapid breakdown in the environment. For this reason, they are preferred over organohalides for insecticide/pesticide use. However, frequent use of OP compounds in agricultural lands has resulted in their presence as residuals in crops, livestock, and poultry products and has further led to their migration into aquifers.6 Typical pesticide concentrations that flow into aqueous waste range from
2 7 10,000 to 1 ppm. • Unfortunately, although these OP compounds are considered safer than organohalides, they are highly toxic to humans and in some cases their
degradation products ( degraded via microbial or environmental processes) have the potential to be more toxic with chronic exposure. OP compounds are extremely
4 dangerous to human health; they are powerful inhibitors of esterase enzymes. They are efficiently absorbed by inhalation, ingestion, and skin penetration. Each year OPs poison thousands of humans across the world, causing hundreds of deaths. In fact, in
1994, an estimated 74,000 children were involved in common household pesticid� related poisoning or exposures in the United States. 1
OP pesticides are used to control moths, ants, cockroaches, fleas, termites, fruitflies, caterpillars and ticks, only to name a few. Although these OP pesticides are sprayed in agricultural lands with the aim of killing or slowing down the growth of these undesirable harmful pests and insects, however, only 1 % reaches its target, 1 while the rest enters the environment. Pesticides can be influenced by a number of processes once it enters into the environment. While many OP pesticides can degrade via microbial or environmental processes, some of the pesticides could get consumed by organisms, or they could leach into ground water. Once a pesticide reaches the ground water it can persist for considerable periods of time. In ground water, there is little sunlight exposure which slows down the degradation of OP pesticides. Thus these pesticides pose potential risks to human health. Figure 1 shows the possible routes of environmental exposure of organophosphorus pesticides to humans and wildlife.8
5 ...... ' ...... ·-' ....' .. '-· ' ...... : fish
Figure 1 Schematic representation of the possible routes of environmental exposure of organophosphurus pesticides to humans and wildlife. 8
1.2.2 Structures of Organophosphorus Compounds
Organophosphorus pesticides (OP) are synthetic and are usually esters, amides, or thiol derivatives of phosphoric, phosphonic, phosphorothioic, or phosphonothioic acids. There are over 100 organophosphorus compounds currently in the market, representing a variety of chemical, physical, and biological properties. As the name indicates, all OP pesticides have a central phosphorus atom, with either double bonded oxygen (P=O), or a double bonded sulfur atom (P=S). A P=O pesticide is called as an oxon pesticide, and the P=S is termed as a thion pesticide.
6 OPs have the following general structure:
Cl The main classes of OPs are:
• Phosphates [R R2 = O]; 1, Cl e.g. Chlorfenvinphos, Dichlorovos Chlorfenvinphos(E,Z)
• Thiophoaphates (Phosphorothioates)
[R2 = N, S, or O];
e.g. Fenthion, Parathion, Diazinon
• Dithiophosphates(Phosphorodithioates) Fenarniphos
[R 1, R2 = S]; e.g. Ethion, Malathion, Disulfoton
• Phosphonates [P-C bond]; CH O- OCH2 3 e.g. Trichlorfon r C l'lil-I H(CH3)z • Phosphoramides Isophenphos [ containing NH2 as R group and O as R 1, R2];
e.g. Fenamiphos, Isophenphos Figure 2 Examples of structures of main classes of OP compounds.
Organophosphorus compounds include a number of nerve gas agents as well as agricultural pesticides and insecticides. As mentioned before, agricultural pesticides
7 or organophosphorus (OP) compounds constitute the most widely used insecticides
available today. Common examples of OP pesticides are shown in Figure 3.
CI Cl �o-,-o---c, -o-l-o�Br 6 0 I . "=<- CI � Dichlorofenthion Bromophos methyl Cl s "-o-!-o--n, \_o-1-o�NoI � 2 Cl - � Bromophos ethyl 9" Parathion ethyl s s \_ II � .,,..-...._ -o-t-o-Q-No O-P-S S� --... - 2 6 1 Para th ion methyl � Phorate
s \_ S N==f- \_o- -o _!�c1 t )-J 0 __ O-i- � CI � Chlorpyrifos � Diazinon
0 -o-1-o�NoI � 2 \_o-1-o�No - I � 2 1 Paraoxon methyl lParaoxon - ethyl Figure 3 Structures of common organophosphorus pesticides.
1.2.3 Health Hazards fromOrganophosphoru s Pesticides
The organophosphorus pesticides are highly neurotoxic to human body. They
are strong inhibitors of cholinesterase enzymes, such as, acetylcholinesterase,
8 butylcholineesterase, and pseudocholinesterase which are neurotransmitters responsible for nerve transmission. The enzymes are inhibited by binding the OP compound which, upon hydrolysis leaves a stable phosphorylated and largely unreacted enzyme. This inhibition results in the accumulation of acetylcholine at the neuron/neuron and neuron/muscle junctions or synapses. The first indication of insecticidal activity among OP compounds was foundin 1930, but the firstcompound of this type, HETP (an impure mixture containing tetraethylpyrophosphate as the
9 active ingredient) was not used as an agricultural insecticide until 1942.
0 0 Eto 1 /oEt ' 1 1 P-0-P1 EtO/ 'OEt Figure 4 Structure ofHETP.
Since the first introduction oh HETP, the number of organophosphorus pesticides has risen to hundred, and the most toxic ones are shown in table 1.2 with their mammalian toxicity levels.
9 Organophosphorus pesticides Mammalian toxicity (rat) LDso mgkg-1
Dipterex 600
Tetraethyl pyrophosphate 1.12
Schradan 20
Dimefox 7
Malathion 1000
Diazinon 100
Parathion 6
Paraoxon 3
Systox 1-30
Table 2 List of the most toxic OP pesticides with their mammalian toxicities.9
1.2.4 Mechanism of Inhibition of Acetylcholinesterase9
Nerve fibers have an electric potential of about 100 mV across their outer wall, and when a nerve gets stimulated, the sign of this potential is momentarily reversed over a small area of the nerve fiber. This small area of reversed potential now travels rapidly along the nerve fiber until it reaches the nerve endings, where it stimulates the release of acetylcholine. If this nerve fiber is now considered to supply impulses to a muscle, the acetylcholine being produced by stimulation of the nerve diffusesinto the muscle surface, and causes a change in electrical potential leading to a contraction.
On arriving at the muscle surface, acetylcholinesterase hydrolyses the ester linkages
10 of acetylcholine which stops the stimulation allowing the muscle to relax. Before another muscle contraction talces place, further nerve stimulation and acetylcholine release is required. However, OP pesticides are anti-cholinesterases which interfere with the process of acetylcholine hydrolysis by deactivating the enzyme. Since the hydrolysis is inhibited, acetylcholine can only leave the area of the muscle by diffusion. On a second stimulation, consequent release of acetylcholine occurs which arrives at the muscle surface where the original acetylcholine is still present. This leads to complete confusion of cause and effect, and the muscle being stimulated undergoes continuous stimulation, a condition named tetanus. 9 Inhibition of acetylcholinesterase is a progressive process depending not only on the concentration of organophosphate concentration, but also on the time of exposure. 10 The sequence of symptoms varies with the route of exposure. Inhalation of the vapor shows respiratory symptoms first, while ingestion shows gastrointestinal problems at first.
II II AChE-OH + _J__ AChE-O-P-OR1
X-1:-0R1 OR2 (A)
Figure 5 (A) Inhibition scheme of acetylcholinesterase (AChE) by organophosphates. (B) Mechanism of inhibition of acetylcholinesterase by OP pesticides.68
11 Figure 5-Continued
r;a�tlcholne
'I{
synaptic cef
i ,watlcho!ine rec�ptor (B)
Symptoms of OP poisoning include rapid twitching of voluntary muscles, followed by paralysis, intense pupil contraction, headache, and abdominal cramps.
Affected individuals are reported to have poor mental health and difficulties with memory and concentration.9 The severity of the symptoms however depends on the degree of acetylcholinesterase inhibition. More severe effects include muscle paralysis which leads to difficulty in breathing, and eventually causes death due to respiratory failure. Because of such adverse effectsof these OPs, their rapid detection in the environment, public places, or workplaces and the monitoring of individual exposures to chemical warfare agents have become increasingly important for homeland security and health protection.
12 The structure of OP pesticides is similar to that of nerve gases, as shown in
Figure 6. These nerve agents are also cholinesterase inhibitors and thus lead to death.
Recently, there has been a major interest in the detection of nerve agents since both soldiers and civilians are at risk of terrorist attacks especially those that may use_ nerve gas agents. In 1995, a terrorist group launched an attack using sarin nerve gas against commuters in the Tokyo subway station. This attack caused 12 deaths and over 5000 injuries.11 The mechanism of inhibition of cholinesterase enzymes by nerve gas agents is similar with OP pesticides.
Sarin LDso i.p. mice 0.6 mg/kg Tabun LD5o i.p. mice 0.6 mg/kg
HaC Cl-la �[ y °" o/ ---s------"I Ha� Cl-la Tetraethyl Pyrophosphate LDso rats oral 1.lmg/kg VX LDso S.C. rabbit 15.4 µg/kg
Figure 6 Structures of some Nerve Gases. 12
Thus, from both a scientific and societal perspective it is of paramount importance to develop a real-time sensor for OP compounds. However, there is a wide range of toxicity in OP compounds, for example the oxon (P=O) form is three orders of magnitude more toxic than the thion (P=S) form and hence is more potent as
13 an inhibitor of acetylcholinesterase. But, thion OP pesticides are relatively more stable, have a long shelf-life and are easily taken up by organisms including humans.
Ideally, sensors that not only detect OP pesticides, but also selectively differentiate between them are in demand.
1.3 Advances in Detection of Organophosphorus Pesticides
Significant advances toward the development of detection methods for OP
13 19 pesticides have been reported in the literature. - Analysis of OPs in environmental and biological samples is routinely conducted using various analytical techniques,
20 19 including NMR , gas, liquid, or thin layer chromatography, and mass spectrometry.
A variety of approaches have been investigated for sensors, including enzymatic
17 13 16 assays, molecular imprinting coupled with luminescence of lanthanides, -
21 23 24 25 colorimetric methods, - surface acoustic waves, • fluorescent organic
26 28 19 18 molecules, - gas-chromatography-mass spectrometry, and interferometry. The most common methods of detection of OP pesticides are the chromatographic methods coupled with different detectors and different types of spectroscopy, immunoassay, and enzyme biosensors based on inhibition of cholinesterase
29 31 activity. - These two common methods of detection of OP compounds, namely, enzyme based sensors and chromatography will be discussed below in further detail.
14 1.3.1 Enzyme Based Detection Methods
To date, a number of sensitive biosensors based on acetylcholinesterase
(AChE) or butyryl cholinesterase (BChE) inhibition have been developed and used
17 29• 32-38 for OP agent detection. · Other enzymes including urease and glucose oxidase have also been used in inhibition-based biosensors for organophosphate
32 33 neurotoxins. • In general, enzyme-based sensors for the detection of organophosphorus compounds can be broadly categorized into two major classes based on the enzyme employed-(!) acetylcholinesterase (AChE) or (2) organophosphorus hydrolase (OPH).
Hydrolysis of acetylcholine by AChE produces one proton per substrate molecule resulting in an increase in the acidity of the solution. This forms the basis for AChE-based sensors. Rogers et al.37 used a pH-sensitive fluorescent dye, consisting of AChE linked to the pH-sensitive fluorescein isothiocyanate (FITC). The enzyme-dye adduct was immobilized on a quartz fiber which was attached to a fluorescence spectrometer. In the absence of an OP compound, the labeled AChE was able to hydrolyze acetylcholine leading to the decrease of pH which resulted in the reduction of the FITC fluorescence intensity due to interruption of the fluorophore's conjugation upon protonation (Figure 7). However, in the presence of diisopropylfluorophosphate (DFP) and subsequently acetylcholine, it was observed that 90% of the enzyme activity has lost, which was quantified by a less pronounced reduction of the fluorescence intensity. This biosensor was found to be very sensitive
(capable of detecting paraoxon in the nM range when exposed to the solution
15 containing the analyte for ten minutes), and it demonstrated some selectivity toward differentOP compounds.
H,("•ro,r�-.;O ✓'-..:,,◊> ...... ;;r,) J. ,,,, .vCO, 1; -.,,,, I·
C s
10�· ,,.,o,.,,,.,.,orl! �-- ,P'• ....:_.) ...,_) )-. ... co; ,, . ...COii C '\(' �) N �) t C s s l'teutral Anion
:'\
4 n pH 8 1 □ 12
Figure 7 (A) Structure of fluorescein isothiocyanate (FITC) at differentpH, (B) its relative fluorescence intensity at selected pH values.38
The second family of biosensors utilizes OPH as the enzymatic sensor for the detection of OP compounds. The mode of action of OPH is different from AChE; it catalytically hydrolyses the organophosphate instead of covalently binding to it. Thus, instead of measuring the enzyme inhibition, detection methods involving OPH allow for a more direct measurement of OP compounds. Nowadays, OPH is widely used as a CWA biosensor because of the ability of its catalytic site to hydrolyze a wide range
11 38 of compounds containing P-O, P-F, P-S, or P-CN bonds. • Hydrolysis of the OP compounds lead to the stoichiometric production of two protons which can be
16 monitored and directly correlated to the amount of OP substrate present. For instance,
36 Cao et al. labeled OPH with FITC and deposited this material onto silanized quartz slides in the form of Langmuir-Blodgett films to create organized monolayers of the enzyme-based sensors. It was demonstrated that this OPH based enzyme sensor showed enhanced sensitivity, detecting the analyte at nM concentrations.
\II ,:a B Zn1' H OH � 0 EIO-f;'-O-0- � N01 11 0-P,O ::.., A NO, Ot EtO "-0- OE1
\ I ' B Zn' 0" H O -P -OEl + 0- � A NO 0 t -0-
Figure 8 Mechanism for the hydrolysis of paraoxon by OPH.
In summary, enzyme based sensors are very sensitive and both selective in their approach to detect organophosphorus compounds. Furthermore, OPH based enzyme sensors offer distinct advantages over AChE-based systems. While these approaches have been significant toward OP detection, the inhibition-based biosensors suffer from three drawbacks; (1) the enzymes easily lose activity in the event of environmental or handling factors, therefore these enzymes may provide
39 40 false positive signals, ' (2) the sensors require baseline testing prior to sample . application and lengthy incubation times to allow enzyme-analyte interaction, and (3) due to the irreversible nature of cholinesterase enzyme inhibition, inhibition-based sensors cannot be reused without regeneration of enzyme activity. In addition, the lifetimeof these sensors is limited by the degradation of enzyme.
17 1.3.2 Chromatographic Detection Methods
Analysis of OP pesticides in the environmental and biological samples is routinely carried out using chromatographic methods, such as gas or liquid chromatography coupled with mass spectrometry. Gas chromatography (GC) is usually the method of detection for vapor phase OP compounds. GC was the first method to be used foranalysis of OP compounds e.g. nerve agents in the laboratory, hence early references date back to the 1960s.41 Kovats retention devices are still important tools in GC for screening the presence of nerve gases.41 Hancock and
Peters investigated the use of retention index monitoring of nerve gas agents in environmental samples by thermal desorption GC in conjunction with simultaneous flame ionization detection (FID) and flame photometric detection (FPD).42 Besides the use of retention indices in combination with different types of GC columns,
Kaipainen et al. investigated the use of different detectors to support identification of various OP compounds.43 Recently, Marx et al. have developed thin films of a molecularly imprinted sol-gel polymer with specific binding sites forparathion. 44 The binding of parathion to the imprinted films was investigated by steady-state experiments with analysis by GC-FPD and cyclic voltammetry. Several new developments in GC are being reported, such as, high-speed GC,45 comprehensive
GC-GC,46 large volume sample introduction by on-column injection or programmed
41 temperature vaporizing (PTV) solvent split injection and new detection methods •
However; inspite of being very sensitive and selective, these high-speed and high
resolution GCs can require up to 30 min for complete separation and detection of a
18 complex mixture of OP compounds which limits their application under rapid field detection.46
Liquid chromatography (LC) is a separation technique with many modes of operation - reversed-phase, normal-phase, ion-exchange, and ion pair and size exclusion, all of which are able to separate essentially all compounds that are soluble in a conventional solvent or solvent mixture, with thermolability, polarity and/or volatility not playing a major role. Thus, LC is the choice of detection method for OP compounds which are not volatile and thermally labile. However, high pressures and small particle sizes limit column length and, therefore, the ultimate resolving power possible by LC.46 Also, another drawback of LC is the lack of detectors matching selectivity and sensitivity of GC detection systems.41 These drawbacks demonstrate the application of GC on OP compound even if aqueous sample matrices are involved.
Although chromatographic methods provide reliable information regarding OP pesticides, such analysis is generally performed at centralized laboratories which require extensive labor and analytical sources, and often results in a lengthy turnaround time. These disadvantages limit the applications of chromatographic methods primarily to laboratory settings and prohibit their use for rapid analysis under field conditions.
19 1.4 The Need
From a practical agricultural viewpoint, the approaches described above have limitations such as low sensitivity, lack of portability, limited selectivity, difficulties in real-time monitoring, and operational complexity. In addition, in most cases, these tests are qualitative or semiqualitative and show false positive and negative results.6
Both soil and water are likely to contain mixtures of OP pesticides due to
6 heavy urban and rural use of these compounds. Unfortunately, there have been no reports to demonstrate molecular sensors that differentiate selectively between OP pesticides. The broad range in toxicity levels of OP pesticide necessitates new methods to discriminate between various OP pesticides.
A key toward practical analytical chemistry is to achieve portability and continuous on-site monitoring of a wide range of analytes which is especially necessary in environmental detection. To meet the requirements of rapid warning and field deployment, more compact low cost instruments, coupled to smaller sensing probes are highly desirable for facilitating the task of on-site monitoring of OP
6 compounds. This goal can be achieved either by miniaturizing established analytical apparatus, for example, separation techniques and spectrometry, as is, for example done in "lab on a chip approaches", well known examples being the miniature DNA analyzers based on capillary electrophoresis which speeded up the identification of the human genome.47 The second approach can be made by development of chemical sensors, i.e. miniaturized• measuring devices optimized to interact with a specified analyte.48
20 1.5 Our Project
Here, we show a class of heterocyclic molecular and nanomaterials as sensors to detect OP pesticides and distinguish between them, based on the pesticide structure. As mentioned previously, organophosphorus pesticides have been extensively used for agricultural purposes for more than 40 years. There are about
200 different compounds on the market, accounting for 45% of the registered pesticides in the USA alone.37 The OP pesticides that will be investigated for the proposed project are shown in Figure 1. All the pesticides that have been chosen for the present case are OP thion insecticides. The reason behind selecting these four is that they are some of the most commonly used OP pesticides. The properties of each of the chosen OP pesticides will be discussed in furtherdetail in the next section.
0
s s /'---.o 0� /'--... /'---.o-�-s s-�-o� 0 s I I s-._ � o � Ethion °� Malathion H3CO/ '----...OCH3
/"--o-�-o I '---.../0 Parathion Fenthion
figure 9 Structures of the organophosphorus pesticides investigated for the project.
21 OP Pesticide Solubility at 20 Vapor Pressure t11 Mammalian
° ° to 25 C (mg/L) at 20 to 25 C (days) toxicity (rat)
LDsomg/kg
Ethion 2 2.0*104 150 100
Malathion 145 5.3*10-.s 1 1000
Fenthion 4.2 7.4*10-" 34 180
Parathion 11 8.9*10-" 3.4 6
Table 3 Physicochemical Properties of the Selected Organophosphorus Pesticides.
From (1) Roger Jeannot and Thierry Dagnac, Organophosphorus Compounds in Water, Soils, Waste, and Air. Chromatographic Analysis of the Environment. Third Edition. (2) BJ. Walker,
Organophosphorus chemistry.
Ethion: This OP pesticide is used to kill aphids, mites, scales, thrips, and maggots. It is also used on a wide variety of food, fiber, greenhouse crops, lawns, and turf.49
Ethion is highly toxic by inhalation, dermal exposure, and ingestion. It is almost non volatile at room temperatures, but when sprayed it can be easily inhaled. The common symptoms of ethion intake are runny nose, irritation to the eye, and a sensation of tightness in the chest. High environmental temperatures or exposure of ethion to visible or UV light may enhance its toxicity.49
Malathion: It is a non-systemic, wide-spectrum OP insecticide. It is one of the earliest OP insecticides developed (introduced in 1950). Malathion is used for the
22 control of sucking and chewing insects on fruits and vegetable, and also applied to control mosquitoes, flies, and household insects.49 It is an OP pesticide with moderate toxicity (LD50 value of 1000 mg/kg in rats). Symptoms of acute exposure to malathion include numbness, incoordination, headache, dizziness, tremor, nausea, and. difficulty in breathing.49
Fenthion: This compound is a widely used insecticide, especially in orchards, and is also frequently used in environmental samples.49 It is an example on an environmental contaminant for which degradation processes are important. The primary degradation products of fenthion are fenthion sulfone, fenthion sulfoxide, fenoxon, fenoxon sulfoxide, and fenoxon sulfone, which are formed through a variety of biological and abiotic pathways. It is an effective pesticide being moderately toxic to mammals, but highly toxic to birds. Symptoms include irritation in eyes and mucus membranes, and difficultyin breathing.49
Parathion: This OP is widely recognized as the cheapest and most effective broad spectrum insecticide.49 Large quantities of parathion are used to protect crop plant roots from soil insects, such as rootworms and wireworms. Its popularity exists primarily due to its high insect mortality rate. It is an aromatic type OP, whose chemical name is O,O-diethyl O-p-nitrophenyl phosphorothioate. Parathion is slightly soluble in water, but completely miscible with many organic solvents. It is a dark, brown liquid with characteristic odor. It can be readily oxidized to its oxon derivative, paraoxon, which is more toxic than the parent compound.
23 Relative toxicity of Parathion: Parathion was the first OP to appear on the market in
1944,50 and is found to be 70 times as toxic as DDT, and hence is capable of producing severe toxic effects and deaths in animals and man. The LD50 value for
Parathion as tested against rats is 6 mg/kg. Parathion can enter human body by swallowing and breathing of mists, dusts or vapors or by absorption through skin.
Pharmacological studies have shown that parathion can perform dual effect after entering into human body: a stimulation of the parasympathetic nervous system and anticholinesterase activity. Inactivation of acetylcholinesterase enzyme due to the presence of Parathion results in accumulation of acetylcholinesterase at the motor end plates of the parasympathetic and the voluntary nervous system, and in the ganglia of the sympathetics. Symptoms include giddiness, headache, nausea, vomiting, diarrhea, muscular fasciculation, and respiratory failure.5
1.5.1 Selective Colorimetric and Electrochemical Detection of Organophosphorus Based Pesticides
An alternative to classical methods of OP pesticides detection is the design of optical detectors, i.e. colorimetric or fluorimetric chemosensors or reagents. One of the most convenient and simple means of chemical detection is the generation of an optical event, i.e. changes in absorption or emission bands in the presence of the target analyte. Optical outputs are being used extensively in recent years for the development of chemosensors for ion or neutral molecule recognition and sensing based on supramolecular concepts.51 Unfortunately, although the utility of optical
24 detection are becoming increasingly appreciated in terms of both qualitative and quantitative analysis, the number of optical sensors available at present for OP compounds detection is quite limited. Of particular interest in this regard are colorimetric OP sensors, species that would allow the so-called naked-eye detection of OP pesticides without having to resort to any sophisticated spectroscopic instrumentation. Currently, there is a surge of interest in the development of tailor made sensor molecules, able to act as chromogenic sensors for a specific analyte. A selective sensor molecule should essentially have a receptor component specific for a selected analyte and a signaling unit capable of translating the analyte-binding induced changes into an output signal.52 The binding event of the analyte and the sensor moiety occurs through intramolecular charge transfer processes which lead to a change of color visible by eye. Materials capable of reversible OP pesticides induced selective changes in color are in great demand as they require little or no instrumentation for practical use. Unfortunately, to our best knowledge, there have been no reports of a selective colorimetric sensor for OP pesticides detection. As mentioned previously, since the toxicity of organophosphates varies by structure, it is important to develop materials that can detect and discriminate between them easily and in real-time. Additionally, materials or techniques that provide independent dual signals are ideal in terms of minimization of false-positive signals. If one of these signals employs an optical technique (i.e. colorimetric or fluorescence) as a signal output, it allows rapid screening and easy user access.
Lately, some innovative methods of detection of OP compounds based on optical methods have been reported in the literature by R.S. Pilato,26 T. M. Swager,28
25 and J.R. Rebek.53 The first fluorescent chemosensor for detection of OP compounds was reported by Pilato et al where a series of non-emissive platinum 1,2-enedithiolate complexes with an appended primary alcohol were synthesized. Upon addition of electrophilic OP analyte to this compound and an activation agent (triazole) in dichloromethane, the alcohol was converted into a phosphate ester, which arranges by intramolecular ring closure reaction to form a fluorescent cyclic product. Although the method was found to be very effective in detection of a variety of nerve agents, the limitation of this work was that the fluorescence of the cyclic platinum complexes was quenched by oxygen, and hence the reactions needed to be performed in ambient conditions which restrict its application in real field purposes.
____ ...._..
26 Figure 10 Mechanism of the chemically reactive sensor developed by Pilato et al.
Swager and his co-workers developed a series of thienylpyridyl and phenylpyridyl systems which undergo intramolecular cyclization reactions upon exposure to nerve agents resulting in a bathochromic shifts in absorption and fluorescence spectra. The fluorescence color changes were observed using a UV lamp under ambient atmosphere. These sensors were found to be both sensitive and
26 selective to chemical warfare agents detection showing a complete response to 10 ppm DFP vapor within five minutes.
FloNsent Ch.ica! Warae .lndlclatots
OY •YX xiOA�
Figure 11 The mechanistic scheme of the chemosensor developed by Swager et. al.28
Rebek et ai53 also did similar work on fluorescent sensors for nerve agents where he examined a series of pyrene based compounds as the fluorescent receptor.
These sensors utilized a similar reaction mechanism designed by Pilato and Swager, however the mechanism in this case was based on the suppression of a photoinduced electon transfer (PET) process to trigger a fluorescent signal. Saturated aliphatic chains ranging fromone to fourmethylene units were employed to determine how the spacer linking the fluorophore and the amine affected the efficiency of the sensors.
Pyrene was used as the fluorophore since it can accept electrons from tertiary amines in PET processes. Upon binding to DCP, the sensors resulted in a significant increase in fluorescent intensity which could be observed visually using a handheld UV lamp.
The sensor displayed practically instantaneous (5 seconds) fluorescence upon exposure to vapor comprising 10 ppm of DCP.
27 hvl hv2 hvl hv.2 \I Flurohore \
0 II � Qp x·�'OR i+O' fOR A' cycliati
Fluoresnce Stro<,g Oueoelle Fluorescence
Figure 12 The chemically reactive sensor reported by Rebek et. al.53
Recently in 2007, Simonian et. al54 reported a fluorescence based sensor for
OP pesticides. They have used a new sensor Coumarinl which is a competitive inhibitor of organophosphorus hydrolase (OPH). Coumarinl in the presence of p nitrophenol substituent organophonates leads to fluorescence quenching due to fluorescence resonance energy transfer (FRET). However, the drawback of this method is that it is applicable only to nitrophenyl substituted pesticides like methyl parathion and fenitrothion.
Figure 13 Coumarin 1, the fluorescent compound and competitive inhibitor as reported by Simonian et. al.54
Here we show a series of four heterocyclic sensors designed not only for detection of OPS, but also to distinguish them, based on the pesticides structure. The
28 sensors display three significant features that make them ideal for this purpose and the hypothesis of this work is driven by these three features:
1) the highly conjugated structure leads to high absorbance of the sensor.
2) the nitrogen atoms within the structure are nucleophilic, and are capable of binding to the electrophilic phosphorus atoms present in the OP pesticides.
3) the nitrogens being directly fused with the chromophore, allow redox-active behavior that could be altered upon OP binding.
1.5.2 Application of Nanoscale Materials as Sensors toward Detection of Organophosphorus Based Pesticides
Metal nanoparticles are very attractive due to their size- and shape- dependent properties. The presence of extraordinary properties in metal nanoparticles arising from their size-dependent properties has been the area of intense research and has promoted a great deal of excitement since the last few decades. Interestingly, these properties are not present in the bulk. As the size of the particle decreases to the 1-
100 nm range, it is well known that the electronic, optical, catalytic, and
55 56 thermodynamic properties of metal nanoparticles deviate from bulk properties. •
This has led to the application of metal nanoparticles into various functions as chemical catalysts, sensors, adsorbents, biological stains, and elements of novel nanometer scale optical, electrical, and magnetic devices.57 Specifically, nanoparticles of silver, gold, and copper show distinct and well-defined plasmon absorption in the visible spectrum. As these noble metals are reduced in size to tens of nanometers, a
29 new very strong absorption is observed resulting fromthe collective oscillation of the electrons in the conduction band from one surface of the particle to another. This oscillation has a frequency that is able to absorb light in the visible range, which is widely known as the surface plasmon absorption.58 This absorption is strong enougll to give rise to vivid characteristic color to the metal nanoparticles. For, example, the gold nanoparticles give rise to a brilliant rose color as the size is reduced to the nanometer regime.
Lately, metal nanoparticles, especially gold nanoparticles have emerged as excellent colorimetric reporters due to their high extinction coefficients59 (the molar absorptivity for the 8 and l3 nm-diameter gold particles was reported to be 7.5x107
M-1 cm-1 at 520 nm and 4.7x109 M-1 cm-1 at 524 nm, respectively)60 which is several orders of magnitude higher than organic dyes and because the transition of the nanoparticles from dispersion to aggregation exhibits a distinct change in color.60
These properties have led to considerable potential of gold nanoparticles to be applicable as colorimetric reporters of ions and molecules as well as macromolecules.
The wide variation of optical properties of gold nanoparticles with particle size, particle-particle distance, and the dielectric properties of the surrounding medium due to surface plasmon resonance enables the construction of simple but sensitive sensors for various analytes. Attention has recently focused on functionalizing gold nanoparticles with molecular recognition components for potential sensing applications.61 Gold nanoparticles possess distinct surface plasmon absorption bands which depend on their size and shape, and the color change upon aggregation is due to the coupling of the plasmon absorbances as a result of their proximity to each
30 other. 62 Aggregation of particles results in color changes of the nanoparticle solutions accompanied by a shift in the SPR peak, due to either change in the dielectric constant around the nanoparticles as a result of adsorption of analyte molecules, or due to analyte-induced agglomeration of the nanoparticles. This aggregation of gold nanoparticles induced by analytes has been utilized for detection of DNA,63 several
62 64 65 61 metal ions, • • and anions, For example, Beer et. al have reported the synthesis of a amide-functionalized zinc metalloporphyrin which was self-assembled on to gold nanoparticles to produce a novel anion-selective optical sensing system.61 Figure
14(A) shows the sketch of the anion-sensing porphyrin gold nanoparticle. For
2+ 2 2+ detection of heavy metal ions (Pb , Hg +, Cd ), Kim et al. have successfully employed gold nanoparticles capped with 11-mercaptoundecanoic acid (MUA) which functions as an appropriate metal ion receptor, figure 14(B) showing the schematic . representation. oft h e react10n sch eme. 64
Figure 14 (A) Schematic of the anion-sensing by Au NP functionalizedwith zinc porphyrin. (B) Scheme of the interaction of MUA capped Au NP with heavy metal IOnS.
31 Recently, various research groups have utilized Au NPs for the detection of
OP compounds. However, these methods sufferfrom several drawbacks. Firstly, although some of the methods were found to be very sensitive, however, none of them are able to detect OP pesticides selectively, but are applicable to a broad range of compounds. Secondly, gold surfaces had to be functionalized in order to bind with the OP compounds which make the methods very tedious and time consuming. In addition, the solvent medium used in most of the cases was organic solvents which prevent advancement of these sensors in practical applications.
Here, we have made use of the aggregation-induced color changes of Au NPs in aqueous solutions as an optical sensor for the detection of organophosphorus based pesticides. The interaction of three different sizes of Au nanoparticles was investigated with ethion, malathion, and fenthion. Citrate is a weak capping ligand.
On the other hand, gold is known to have a high affinity to bond with sulfur atoms because of their soft characters. Thus, when the OP pesticides containing several sulfur atoms come in contact with the gold nanoparticles, the sulfur atoms bind to the
Au surface releasing the capped citrate into the solution. This leads into aggregation of Au nanoparticles resulting in visible color changes which can be readily detected by the naked eye. The novel method of sensing developed by us offer three essential features (i) enables to use Au NPs capped with citrate as sensors for OP detection (ii) the detection was found to be specific with differentOPs based on their structures and
(iii) ensures proper functioning of the sensors in aqueous environment.
32 1.6 Research Objectives and Hypothesis
The research objectives of this work are:
1) Design and synthesize a molecular sensor that undergoes changes m optical properties upon interaction with organophosphorus pesticides.
2) Identify sensor with electrochemical properties that could potentially yield an additional signal output upon OP pesticides interaction, thus minimizing false positives.
3) Development of nanoscale sensors based on gold nanoparticles for detection of organophosphorus pesticides via optical signal transductions, while ensuring proper function in aqueous environments.
4) Optimize sensor characteristics so that the sensors distinguish between various organophosphorus based pesticides.
5) Determination of the detection limits, to ensure that sensors function within the desired range.
The construction of receptors which can selectively recognize and sense organophosphate guest species via macroscopic physical response is a current area of
46 54 64 chemical sensor technology receiving considerable attention. • • The described research is driven by designing and investigating a series of nitrogen containing heterocycles based on stilbene family. The interactions of these compounds were then examined with four different kinds of OP pesticides as mentioned, namely, ethion, malathion, fenthion, and parathion. These molecular sensors have a highly conjugated structure so as to yield high fluorescence quantum yields and absorb in the UV - visible region. The presence of the nucleophilic nitrogen atoms within the structure
33 allows binding to an electrophilic phosphorus atom in the OP pesticides. The directly fusedheteroatom on the chromophore also allows redox-active behavior that could be altered upon OP binding, and therefore producing independentdual signal outputs to minimize false-positive signals.
The second part of our research dealt with the development of nanomaterials as sensors based on gold nanoparticles for the detection of OP pesticides. Three different sizes of Au nanoparticles capped with sodium citrate were synthesized, and their interaction of with ethion, malathion, and fenthion was investigated. The mode of detection was once again based on optical methods. We have made use of the aggregation-induced color changes of Au NPs in aqueous solutions as an optical sensor forthe detection of organophosphorus based pesticides.
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42 CHAPTER 2
SELECTIVE COLORIMETRIC AND ELECTROCHEMICAL DETECTION OF ORGANOPHOSPHORUS BASED PESTICIDES
2.1 Introduction
2.1.1 Chemical Sensors and Molecular Recognition
Over the past two decades, chemical sensors, which are defined as reagents that interact with an analyte with high affinity and yield a measurable signal in response, have developed as viable alternatives to traditional methods of analysis.1
They play a crucial role in the detection of water, oxygen, carbon-dioxide, pH, metal ions, contaminants in waste water, and in the elucidation of cellular mechanisms. An ideal chemical sensor offers the advantage of in situ measurements, and provides information in real-time. It must recognize the analyte with high specificity, and have an affinitycommensurate with the average concentration of the analyte solution.2
Molecular recognition, "the process involving both binding and selection of
3 substrates by a given receptor molecule as well as possibly a specific function" , is opening the way to several desirable goals, such as responsive or intelligent materials, molecular devices, and new sensors.4 The ideas of molecular recognition have been used in applications in biological and material sciences and the emerging field of supramolecular chemistry. Molecular recognition is a question of information storage
43 and read out at the supramolecular level.3 The recognition process involves binding with a purpose, implying a structurally well-defined pattern of intermolecular interactions. 5
Just as there is a field of molecular chemistrybased on the covalent bond, there is a field of supramolecular chemistry, the chemistry of molecular assemblies and of the intermolecular bond.6 Molecular receptors are organic structures, held by covalent bonds, which are able to bind selectively with various ions or molecules. This binding makes use of various intermolecular interactions, namely, electrostatic interactions, hydrogen bonding, Vander Waals forces, short range repulsions, etc. and results to the formation of an assembly of two or more molecules, a supermolecule. The design of the receptor determines which substrate is bound. The substrate-specific synthesis of a supermolecule thus involves organic (regioselectivity; stereospecific) synthesis of a receptor by formation of covalent bonds, followed by one or several binding steps using intermolecular bonds in an arrangement predetermined in the design of the receptor.
The work described herein, uses the concepts of molecular recognition to develop selective receptor molecules for organophosphorus pesticides. The receptors are comprised of aromatic heterocycles with nitrogen atoms that serve as nucleophiles. The binding process involves signal transduction, and we measure this signal as a change in the optical property of the ligand. The processes of molecular recognition are summarized in Figure 15. We are interested in designing chemical sensors that upon binding to the substrate produce an optical signal.
44 II Synthesis...... �\�._ x· II
OP peSticide Sensor- OP complex formation should result in a recognition event
Figure 15 Schematic representation of the process of molecular recogmtlon: a receptor is synthesized with recognition units or binding sites for the substrate. The receptor recognizes the substrate with high specificity and binds to it through intermolecular interactions. The binding process takes place with high recognition resulting in signal transduction.
2.1.2 Advances in Optical Methods of Detection of Organophosphorus Compounds
An alternative to classical methods of OP pesticides detection is the design of optical detectors, i.e. colorimetric or fluorimetric chemosensors or reagents.
Unfortunately, although the utility of optical detection are becoming increasingly appreciated in terms of both qualitative and quantitative analysis, the number of optical sensors available at present for OP compounds detection is quite limited. Of particular interest in this regard are colorimetric OP sensors, species that would allow the so-called naked-eye detection of OP pesticides without resort to any sophisticated spectroscopic instrumentation. A selective sensor molecule should essentially have a
45 receptor component specific for a selected analyte and a signaling unit capable of
translating the analyte-binding induced changes into an output signal.8 Materials
capable of reversible OP pesticides induced selective changes in color are in great
demand as they require little or no instrumentation for practical use. Unfortunately, to our best knowledge, there have been no reports of a selective colorimetric sensor for
OP pesticides detection. Since the toxicity of OP pesticides varies by structure, it is important to develop materials that can detect and discriminate between them easily
and in real-time. Additionally, materials or techniques that provide independent dual signals are ideal since they could minimize false-positive signals. If one of these signals employs an optical technique (i.e. colorimetric or fluorescence) as a signal output, it allows rapid screening and easy user access.
Lately, some innovative methods of detection of OP compounds based on optical methods have been reported in the literature. R.S. Pilato,9 T. M. Swager,10 and
J.R. Rebek11 are worth-mentioning names here. However, all these methods were directed to design optical sensors for chemical warfare agents,and not OP pesticides.
As mentioned before, there have been no reports of sensors which selectively
differentiate between various OP pesticides. Here, we show a heterocyclic sensor
based on the family of stilbenes. The sensor has been designed not only for detection
of OP pesticides, but also for selectively distinguishing them, based on the pesticides
structure. The sensor display three significant features that make it ideal for this
purpose and the hypothesis of this work is driven by these three features:
1) The push-pull stilbene system leads to high molar extinction coefficients due to an
intramolecular charge transfertransitions leading to absorption in the visible region.
46 2) the highly conjugated structure leads to high fluorescence quantum yields.
3) the nitrogen atoms within the structure are nucleophilic, and are capable of binding to the electrophilic phosphorus atoms present in OP pesticides.
4) the nitrogens being directly fused with the chromophore, allow redox-active behavior that could be altered upon OP binding.
Figure 16 shows the structure of the molecular sensor designed by us.
Dimethyl-[ 4-(2-quinolin-2-yl-vinyl)-phenyl]-amine (DQA)
Figure 16 Structure of the molecular sensor.
2.2 Experimental Section
2.2.1 Materials and Instrumentation
Quinaldine (2:90%), benzaldehyde (2:99.5%), dimethylsulfoxide (anhydrous
DMF, 2:99.8% ), potassium tertiarybutoxide (2:97%), lithium hydride (2:95%), ammonium chloride (2:99.5%), hydrochloric acid (2:37%), ethion, malathion, fenthion, and parathion were obtained from Sigma-Aldrich. Tetrabutylammonium hexafluorophosphate (TBAPF6, 2:99%) was obtained from Fluka. The solvents
47 fenthion, and parathion were obtained from Sigma-Aldrich. Tetrabutylammonium hexafluorophosphate (TBAPF6, 2:99%) was obtained from Fluka. The solvents dichloromethane ( anhydrous, 2:99.5%) and acetonitrile (2:99.5%) were obtained from
Sigma-Aldrich. All solvents were of HPLC grade or better and were dried prior to usage. UV-visible absorbance spectra were acquired using a Varian Cary 50 spectrophotometer. Emission spectra were acquired using a Varian Eclipse
1 31 spectrofluorometer. H and P Ni\.1Rwas recorded on a 400 :MHzJEOL spectrometer at room temperature, and deuterated acetonitrile (2:99.5%) was used as the solvent.
2.2.2 Synthesis ofDQA
0 DMF, Potassium II tertiarybutoxide N � Stirred for 6hrs at room temperature # ,/D--/ # N/'----... (DQA)
(Quinaldine) (Benzaldehyde) H20
Scheme 1 Synthesis ofDimethyl-[ 4-(2-quinolin-2-yl-vinyl)-phenyl]-amine (DQA).
Equivalent moles of quinaldine (0.677 ml, 0.005 mole) and p dimethylaminobenzaldehyde (0. 74595 g, 0.005 mole) were measured out in a glass vial. To this mixture, 5 ml of fresh and dry DMF was added, and shaken to dissolve completely. To an oven-dried 25 ml round bottom flak, a magnetic stir bar was added,
48 Lithium hydride (0.099 g, 12.5 mmol) and potassium t-butoxide (0.70131 g, 6.25 mmol) were added to the flask one after another, making sure that the drying tube remains attached after each addition. The reaction mixture was stirred for 5 minutes.
After stirring was done, the mixture previously prepared in the glass vial was added to the round bottom flask, once again making sure that the drying tube was replaced on the flask after the addition. This reaction mixture was then stirred for 24 hours.
About 15 g of ice was taken in a large beaker to which 10 ml of saturated ammonium chloride (NHiCl) was added. The stirred reaction mixture was then poured into the beaker of ice, and 5 ml of saturated ammonium chloride was added to it to dissolve any remaining product in the flask. The mixture in the beaker was stirred forabout 15 minutes, and the resulting solid was collected using a vacuum filter. The solid product was washed with cold water while the vacuum was on, and left in the vacuum to air dry the product overnight. The average yield of this product was 75%. The product
1 1 was characterized by H NMR and UV-visible spectra. H NMR (CD3CN, 400 MHz)
8: 8.1890-8.1679 (d, lH, J = 8.44 Hz), 7.9527-7.9316 (d, lH, J = 8.44 Hz), 7.8519-
7.8309 (d, lH, J = 8.4 Hz), 7.7210-7.6715 (q, lH, J = 19.8 Hz), 7.5680-7.5460 (d, lH,
J = 8.8 Hz), 7.5067-7.4865 (q, lH, J = 8.08 Hz), 7.2017-7.1614 (d, lH, J = 16.12 Hz),
6.8253-6.7639 (d, lH, J = 24.65 Hz), 1.9429 (s, 6H).
2.2.3 UV-visible Absorbance Measurements
A Varian Cary 50 spectrophotometer was used to measure the UV-Visible absorbances of the sensors, as well as OP-induced UV-visible absorbance changes. In
49 each case, an acetonitrile solution of the sensors was freshly prepared at room temperature. The sensor solution was then titrated with a solution of the pesticide. In most cases, neat pesticide was used for the titration experiment. The titrations were done to observe the UV-visible absorbance changes that occurred upon interaction of the sensor and the OP pesticides. UV-visible absorbance measurements were taken immediately following each addition of OP pesticides added. All measurements were taken at room temperature.
2.2.4 Fluorescence Measurements
Varian Cary Eclipse spectrofluorometer was used to measure fluorescence of the sensors as well as OP-induced fluorescence changes using an excitation wavelength of 330 nm. In the case of titration with fenthion, an excitation wavelength of 375 nm was used. In each case, an acetonitrile solution of the sensors was freshly prepared at room temperature. The sensor solution was then titrated with a diluted solution of the pesticide. Fluorescence measurements were taken immediately following each addition of OP pesticides added.
2.2.5 Electrochemical Measurements
Cyclic voltammetry was used to measure redox potentials of the sensor ( ~ 10-
4M) m 10-4 M TBAPFJacetonitrile solutions. A BAS model CV-50W
50 electrochemical workstation was used in a standard three-electrode arrangement consisting of a glassy carbon working electrode, a Pt wire counter electrode, and a
Ag/AgCl reference electrode.
2.2.6 Computational Modeling
All studies were optimized at the B3LYP level of density functional theory.
The standard 6-31+G(d,p) was used. Frequency calculations at the same level of theory have also been performed to confirm that all stationary points were minima
(no imaginary frequencies). To acquire the mapped electrostatic potential, the isosurface values are set to 0.010. All calculations were performed with the Gaussian software package. 23
1 31 2.2.7 H AND P NMR Measurements
All NMR experiments were recorded on a 400 MHz JEOL spectrometer with chemical shifts (ppm) relative to trimethylsilane (TMS).
51 2.3 Results and Discussion
The interactions of DQA with four OP pesticides, ethion, malathion, fenthion, and parathion were investigated.
The absorption spectrum of DQA in acetonitrile shows a peak at 385 run which can be assigned due to intramolecular charge transitions from the dimethylamine nitrogen to the quinaldine nitrogen. 15 Moreover, in acetonitrile medium, DQA emits in the green with Amax = 524 run, as shown in figure 18. The emission intensity is readily observed with the naked eye under UV-light.
52 0.5 385nm 0.4
.0 0.3
.0 0.2
0.1
0 300 350 400 450 500 550 600 Wavelength (nm)
Figure 17 UV -visible absorbance spectrum of DQA in acetonitrile.
160 524nm
120
"iii
C: 80
"iii E w 40
350 400 450 500 550 60 650 700 Wavelength (nm)
Figure 18 Fluorescence spectrum of DQA in acetonitrile.
53 2.3 .1 UV -visible Absorbance Spectroscopy Studies
2.3.1.1 Investigation of the Interaction of DQA with Ethion
A 2.4x10-5 M solution of DQA in acetonitrile solvent was prepared. Changes in the UV-Visible spectrum were monitored, as neat ethion (Aldrich) was added to
3 the DQA solution in 2 µL (2.lxl0- M) increments. As shown in Figure 19, addition of ethion resulted in the quenching of the 385 nm peak of the sensor, along with the formation of two new peaks at 500 nm and 325 nm indicating the formationof a new species upon complexation of the sensor and ethion. Also, the appearance of two isosbestic points at 340 nm and 425 nm in the titration profile indicates that two species are coexisting at the equilibrium with a 1: 1 stoichiometric ratio. The formation of the new species is also evident from a change in color of the solutions, the yellow color of the sensor solution changes results in a bright red colored solution upon addition of ethion.
54 1.2
1
0.8 a> (J
,e 0.6
0.4
0.2
300 400 500 600 700 Wavelength (nm)
Figure 19 Changes in the UV-visible absorbance titration profile of DQA in the presence of increasing concentrations of ethion. From top to bottom, [ethion] = 0, 2.1, 4.2, 6.3, 8.5, 10.6, 12.7, 14.8, 16.9, 19.0, 23.3, and 25.4 mM. Inset showing the color change of DQA upon addition of ethion.
2.3.1.2 Investigation of the Interaction of DQA with Malathion
A 2.4x10-5 M solution of DQA in acetonitrile solvent was prepared. Changes in the UV-visible spectrum were monitored, as neat malathion (Aldrich) was added to the DQA solution in 2 µL (2.5x10-3 M) increments. As shown in Figure 20, addition resulted in the quenching of the 385 nm peak of the sensor, along with the formation of two new peaks at 500 nm and 320 nm indicating the formation of a new species upon complexation of the sensor with malathion. Also, the appearance of two isosbestic points at 335 nm and 420 nm in the titration profile indicates that two
55 species coexist at the equilibrium with a 1: 1 stoichiometric ratio. The formation of the
new species is also evident from a change in color of .the solutions, the yellow color
of the sensor solution changes results in an orange colored solution upon addition of
malathion.
�06
� C 0.4 m .c 0 .cIll <(
0.2
oL----��;::;:;� 300 400 500 600 700 Wavelength (nm)
Figure 20 Changes in the UV-visible absorbance titration profile of DQA in the presence of increasing concentrations of malathion. From top to bottom, [malathion] = 0, 2.5, 4.9, 7.4, 9.9, 12.3, 14.8, 17.3, 19.7, 22.2, 24.8, 27.3, 29.7, 32.3, 34.7, 37.2, and 39.7 mM. Inset showing the color change of DQA upon addition of malathion.
56 2.3.1.3 Investigation of the Interaction of DQA with Fenthion
A 2.4x10-5 M solution of DQA in acetonitrile solvent was prepared. Changes in the UV-visible spectrum were monitored, as neat fenthion (Aldrich) was added to
3 the DQA solution in 2µL (3.0xl0- M) increments. As shown in Figure 21, at the end of the titration, the absorbance intensity was relatively similar to the original absorbance of DQA. The yellow colored DQA still remained yellow after fenthion addition. This indicates the lack of any complex formation between the sensor and fenthion.
0.4
uQ) C ftl .0 0 Cl) . 0.2 c(
0 +---�--�--�--==�"'T------r-----.-----r--� 300 350 400 450 500 550 60 650 700 Wavelength (nm)
Figure 21 Changes in the UV-visible absorbance titration profile of DQA in the presence of increasing concentrations of fenthion. From top to bottom, [fenthion] = 0, 3.0, 6.0, 9.0, 12.0, 15.0, 18.0, and 21.0 mM. Inset showing the color change of DQA upon addition of fenthion.
57 2.3.1.4 Investigation of the Interaction of DQA with Parathion
A 2.4x10-5 M solution of DQA in acetonitrile solvent was prepared. Changes in the UV-visible spectrum were monitored, as neat parathion (Aldrich) was added to
3 the DQA solution in 2 µL (2.9x10- M) increments. As shown in Figure 22, addition resulted in the formation of a new peak at 505 nm indicating the formation of a new species. The new peak continually grows as more parathion is introduced into the solution. The formation of the new species is also evident from a change in color of the solutions, the yellow color of the sensor solution changes results in a yellow orange colored solution upon addition of parathion.
0.6
� 0.4 C C'IS .0
.0"' 0.2
300 400 500 600 700 Wavelength (nm}
Figure 22 Changes in the UV-visible absorbance titration profile of DQA in the presence of increasing concentrations of parathion. From bottom to top, [parathion] = 0, 2.9, 5.8, 8.7, 8.5, 11.6, 14.5, 17.4, 20.3, 23.2, 26.1, 29.0, 31.9, 34.8, 37.7, and 43.5 mM. Inset showing the color change of DQA upon addition of parathion.
58 2.3 .1.5 Summarized Results of the UV -visible Titration Data
Table 2.1 summarizes the results obtained from the UV-visible absorbance spectroscopy measurements. It was found that DQA is very successful in distinguishing all the four OP pesticides resulting in different colored solutions with different Amax values. This is very desirable since no reports for a selective colorimetric sensor for detection of OP pesticides has been reported before. In order to understand the strength of the binding taking place between DQA and the corresponding OP pesticides, binding constants were calculated. It was found that
DQA undergoes strongest binding with ethion, followed by malathion, and fenthion ..1/ �o.,caW.:.c<,
OP Pesticide Color of the >ax fr the Detection
final solution new peaks limits Binding constant (M1)
(nm)
6 4 Ethion Red 500,325 10- M 6.5xl0
6 4 Malathion Light pink 500,320 10- M l.lxl0
Fenthion Yellow None NIA No interaction
6 4 Parathion Yellow-orange 505 10- M 0.2xl0
Table 4 Summary of results forthe interaction of DQA with the OP pesticides.
Furthermore, in order to support the data obtained fromUV-visible absorbance spectroscopy results, further studies were carried on with DQA which will be discussed in the next sections.
59 2.3.2 Fluorescence Spectroscopy
2.3 .1.1 Investigation of the Interaction of DQA with Ethion
Changes in the fluorescence spectra of DQA with the OP pesticides were investigated.
A 2.4x10-s M solution of DQA in acetonitrile solvent was prepared. Changes in the fluorescence spectrum were monitored, as neat Ethion (Aldrich) was added to the DQA solution in 2 µL (2.lxl0-3 M) increments. As shown in Figure 23, addition resulted in the quenching in fluorescence intensity of the 524 nm peak of the sensor.
At the saturation point, the blue emission of DQA was 100% quenched and the solution was colorless. It is possible that the donor (DQA) and the acceptor ( ethion) in this case results in the formation of a new DQA-ethion complex which is non fluorescent.
60 140
120
_100
80 ·u; C
C
40
20
0 390 440 490 540 590 640 690 Wavelength (nm)
Figure 23 Changes in the fluorescence titration profile of DQA in the presence of increasing concentrations of ethion. From top to bottom, [ethion] = 0, 2.1, 4.2, 6.3, 8.4, 10.5, 12.8, and 14.7 mM.
2.3.2.2 Investigation of the Interaction of DQA with Malathion
A 2.4x10-5 M solution of DQA in acetonitrile solvent was prepared, and changes in the fluorescence spectrum was monitored, as neat malathion (Aldrich) was added to the DQA solution in 2 µL (2.5x10-3 M) increments. As shown in Figure 24, addition resulted in the fluorescence quenching of the 524 nm peak of the sensor. At the saturation point, the blue emission of DQA was 100% quenched and the solution was colorless. It is possible that the donor (DQA) and the acceptor (malathion) in this case results in the formation of a new DQA-malathion complex which is non-
61 fluorescent. It is worth mentioning here that it requires much higher concentration of malathion compared to ethion in order to result in 100% quenching, indicating that ethion undergoes a stronger binding with DQA than malathion, which follows our expectation as judged by the binding constant values.
200
180
160
140
� 120
� 100
. 80
60
40
20
0 390 440 490 540 590 640 690 Wavlength (nm)
Figure 24 Changes in the fluorescence titration profile of DQA in the presence of increasing concentrations of malathion. From top to bottom, [malathion] = 0, 2.4, 4.9, 7.4, 9.9, 12.3, 14.8, 17.3, 19.7, 22.2, 27.1, 29.5, 32.1, 34.5, and 37.0 mM.
62 2.3.2.3 Investigation of the Interaction of DQA with Fenthion
A 2.4x10-5 M solution of DQA in acetonitrile solvent was prepared, and changes in the fluorescence spectrum was monitored using an excitation wavelength of 375 nm, as fenthion (diluted in acetonitrile) was added to the DQA solution in 5
µL (7.47x10-5 M) increments. As shown in Figure 25, addition resulted in no significant changes in the fluorescence intensity of DQA indicating that fenthion is non-interactive with DQA in accordance with the UV-visible absorbance data.
500
450
400
350
;- 300
� 250
.E 200
150
100
50
0 400 450 500 550 600 650 Wavelength (nm)
Figure 25 Changes in the fluorescence titration profile of DQA in the presence of increasing concentrations of fenthion. From top to bottom, [fenthion] = 0, 3.0E-4, 6.0E-4, 9.0E-4, 12.0E-4, and 15.0E-4 M.
63 2.3.2.4 Investigation of the Interaction of DQA with Parathion
A 2.4x10- 5 M solution of DQA in acetonitrile solvent was prepared, and changes in the Fluorescence spectrum was monitored, as diluted parathion (diluted in acetonitrile) was added to the DQA solution in 5 µL (3.61xl0-5 M) increments. As shown in Figure 26, addition resulted in the quenching in fluorescence intensity of the
524 nm peak of the sensor. At the saturation point, the blue emission of DQA was
100% quenched and the solution was colorless. The binding of a nitro (-N02) group
1 to a fluorophore is well-known to quench the emission of most fluorophores. The observed quench in fluorescence may be attributed to DQA-parathion complex formation, whereby the -N02 group results in fluorescence quenching of DQA.
200
160
; ai ";: 120 := ell C
80
40
390 440 490 540 590 640 Wavelengh (nm)
Figure 26 Changes in the fluorescence titration profile of DQA in the presence of increasing concentrations of parathion. From top to bottom, [parathion] = 0, 3.6E-5, 7.2E-5, l.lE-4, l.4E-4, l.8E-4, 2.2E-4, 2.9E-4, 3.6E-4, 4.3E-4, 5.lE-4, 5.8E-34, 7.2E-4, 8.7E-4, l.0E-3, l.2E-3, 1.3E-3, 1.4E-3, l.7E-3, 2.0E-3, and 2.3E-3 M.
64 2.3.3 Electrochemical Measurements
While the UV-visible absorbance spectroscopy measurements provided significant results and showed the selectivity toward OP pesticides, it was important to determine whether an alternative signal output could additionally be used to demonstrate whether binding had occurred. From a general viewpoint, sensors that provide dual independent signal outputs are advantageous as they minimize the risk of false-positive signals. Electrochemical sensors are advantageous for several applications as they can be developed into miniaturized devices and used for field based and in-situ environmental monitoring. 13 These type of sensors are concerned with the interplay between electricity and chemistry, namely the measurements of electrical quantities, such as current, potential or charge and their relationship to chemical parameters where the applied potential between a reference and a working electrode causes the oxidation or reduction of an electroactive species. 13
To obtain a second 'validating' signal output, the binding of the OP pesticides by the sensors were investigated by cyclic voltammetry (CV). DQA was found to be electroactive, being able to get oxidized and reduced. Figure 27 shows the example of
the cyclic voltammogram obtained for DQA (8.75x10·8 M) alone in acetonitrile
medium (E 112 = -845 mV vs. Ag/AgCl). Therefore, we investigated whether the new
complexes formed upon binding of the differentOP pesticides with DQA would yield
different electrochemical properties in comparison to the parent compound alone.
65 5.00E-05
4.00E-05
3.00E-05
2.00E-05
0 1.00E-05
0.00E+00
-1.00E-05
-2.00E-05 Potential (mV) vs. Ag/AgCI
Figure 27 Cyclic voltammogram of 8.75x10-4 M DQA alone in acetonitrile (working electrode: glassy carbon, counter electrode: Pt wire).
As discussed before, DQA was found to yield distinct changes in the colors of the solutions with ethion, malathion, and parathion, but it did not show any interaction with fenthion. CV data also did not show any changes in the redox activity of DQA upon addition of fenthion, indicating once again that fenthion is not capable of undergoing binding with DQA in the ground state. However, addition of ethion did not show any cathodic or anodic peaks in the spectrum which might be due to the fact that the DQA/ethion complex is non-electroactive. But addition of malathion resulted in the formation of DQA/malathion complex which was accompanied by a specific change in redox potential, as well as formation of a new redox peak (DQA/malathion complex: E112 = -1498 mV vs. Ag/AgCl, E112 = -870 mV vs. Ag/AgCl).
66 5.00E05 ------� -DOA -DOA/malathion complex 4.0E05 1------�
3.00E05 < s_ 2.00E05
0 1.0E05
-100 -1200 -1400 -1600 -180 -2
-1.00E05
-2.00E05 �------Potential (mV) vs Ag/AgCI
Figure 28 Changes in redox properties of DQA upon addition of 7x10·4 M malathion.
On the other hand, addition of parathion resulted in the formation of
DQNparathion complex demonstrating significant changes in the redox behavior
(E112 = -1072 mV vs. Ag/AgCl, E112 = -773 mV vs. Ag/AgCl) in comparison to the
parent compound. The reduction waves show cathodic shifts, indicating that the DQA
complex is difficult to reduce. Moreover, addition of parathion to the DQA solution
resulted in a gradual increase in current which can be attributed to the formation of
the DQNparathion complex with a higher diffusion coefficient. Figure 29 shows an
example of the CV of DQA titrated by parathion.
67 1.20E04 -DQA 1.00E04 -DOA/parathion complex 8.00E05
6.00E05
4.00E05
0 2.00E05
O.OOE+OO
-2.00E05 -1500
-4.00E05
-6.00E05
-8.00E05 Potential (mV) vs Ag/AgCI
3 Figure 29 Changes in redox properties of DQA upon addition of 7x10- M parathion.
2.3.4 NMR Studies
1 31 H and P NMR studies were performed before and after titration of DQA with the OP pesticides in order to determine the structures of the new complexes.
DQA and the corresponding OP pesticides were mixed in varying molar ratios in deuterated acetonitrile solvent and changes in the NMR spectra were recorded.
In the case of 1H NMR, the chemical shifts of the C-H proton in the quinoline ring (Hi, 8 = 8.1890 ppm) and the ethylene proton (H2, 8 = 7.2017 ppm) right next to the nitrogen atom of DQA was found to be sensitive to the addition of ethion. The chemical shifts of these two protons are progressively downfield shifted and remained
68 unchanged upon addition of 1 equivalent of ethion. The final displacement of the protons was 0.0017 and 0.0018 ppm respectively. The downfield shifts can be assigned due to binding of the nitrogen atom in DQA with the phosphorus atom in ethion which creates a shielding effect to these protons changing their surrounding chemical environment. Similar downfield shifts were also observed with the addition of malathion and parathion to DQA, however the chemical shifts in these cases were less prominent compared to the ethion complex. No changes were observed for fenthion-DQA complex, once again demonstrating that fenthion is non-interactive in the ground state.
../ N "-._
Figure 30 Structure of DQA showing the protons susceptible to undergo chemical shifts upon binding with the OP pesticides.
69 c·
b ( a
8.06 8.05 8.04 8.03 8.02 8.01 Nppm
1 5 Figure 31 H NMR shifts in the quinaldine nitrogen of 3.3x10- M of DQA in CD3CN upon addition of ethion in (a) 1:0.4 (b) 1:0.8, and (c) 1:1 molar ratios.
The 31 P NMR of the OP pesticides in acetonitrile was compared with the 31P
NMR of DQA with the corresponding OP pesticides in acetonitrile. Surprisingly, no significant differences were observed. This can be attributed to the fact that chemical shifts of 31P NMR are greatly affectedby the unbalance of the cr-bonds as determined
16 by the electronegativities of the substituents attached to the phosphorus atom. Based on the Pauling electronegativity scale,17 the difference in the electronegativity of nitrogen (3.066), oxygen (3.610), and sulfur (2.589) atoms is not much. It is possible that due to close proximity of these values, the changes in the chemical environment of the phosphorus atom are insignificant when it binds to the nucleophilic nitrogen on
DQA, and hence no significant changes in the 31P NMR were observed.
70 2.4 Investigation on the Binding Sites in DQA
DQA has two nucleophilic sites in its structure- the quinoline and the dimethylamine group, and hence a 1 :2 stoichiometry complex was expected to form upon interaction of DQA with the OP pesticides. Technically both the sites are capable of donating their lone pair of electrons to the electrophilic phosphorus atoms in the OP pesticides structure. Usually, due to presence of the electron donating methyl groups on the dimethylamine nitrogen, this site is expected to be more basic than the quinoline site and hence should have higher tendency to attack the phosphorus atom. However, with the involvement of intramolecular charge transfer
(ICL) in the ground state of DQA, the quinoline site becomes more basic compared to the dimethylamine site.15 Thus the quinoline nitrogen atom is more prone to attack the electon deficient phosphorus atoms of the OP pesticides.
In order to identify the molecular static potential and to determine the nucleophilicity of the two nitrogen atoms, computational calculations on DQA was performed with the Gaussian 03 software package.23 As discussed above, the quinoline nitrogen was expected to be more nucleophilic, and computational studies confirmed this hypothesis. The data in Figure 2.17 shows that the electrostatic potential at the quinoline nitrogen is much higher compared to the dimethylamine nitrogen atom showing that formation of a 1 :2 stoichiometric complex is not very feasible.
71 Figure 32 Molecular electrostatic potential of DQA calculated by Gaussian 03 software package. The scale shown above the structure represents the charge and is an absolute scale.
In addition, 1 H NMR studies were investigated in order to understand whether the computational data translates well with the experimental data. 1 H NMR studies show that chemical shifts of H1 and H2 in DQA progressively downfield shifts with gradual addition of the OP pesticides. However, the downfield shift remains unchanged upon addition of 1 equivalent of the OP pesticides, as shown in figure 33.
This confirms a 1: 1 stoichiometry upon interaction of DQA and the OP pesticides.
72 8.192
8.1915
8.191
E 8.1905 C. C. � 8.19 8.1895
8.189
8.1885 0 0.5 1.5 2 2.5 3 3.5 equivalents
Figure 33 Chemical shifts in H 1 with gradual addition of ethion.
Furthermore, we synthesized an analogue of DQA, 1-quinoli-2-phenethylene
(QPE) without containing the dimethylamine group in order to investigate the role of the two nitrogen binding sites. QPE in acetonitrile gives a colorless solution with an absorbance peak at 320 nm. A 2.lxl0-5 M solution of QPE was prepared in acetonitrile and changes in the UV-visible spectrum were monitored as neat ethion
3 (Aldrich) was added to the QPE solution in 2 µL (2.lxl0- M) increments. With the introduction of ethion into this solution, the 320 nm absorbance peak of QPE gradually decreases, along with the formation and growth of a new peak at 370 nm.
The appearance of an isosbestic point at 345 nm indicates the formation of a new species. However, because of the formation of the new peak at 370 nm (not in the visible range), there was no change in the color of the solution with ethion. This demonstrates that the quinaldine nitrogen is the only binding site with the OP
73 pesticides, but the dimethylamine nitrogen is only involved in the intramolecular charge transfer in the molecule resulting in formation of colors in the visible region.
This concludes that a 1: 1 stoichiometry takes place upon binding of DQA with ethion.
Figure 34 shows the structure of QPE and its UV-visible titration data with ethion.
(A)
0.5
(B) U.4
� 0.3 C Ill -e 5l ct 0.2
0.1
0-t------,------,------, 300 400 500 600 Wavelength (nm)
Figure 34 (A) Structure of QPE, and (B) Changes in the UV-visible absorbance titration profile of QPE with the addition of ethion, inset showing the colors of the solutions before and after titration.
74 2.5 Mechanism of Interaction
The mechanism of interaction of DQA occurs through the nucleophilic attack of the pyridine nitrogen at the electrophilic phosphorus center present in the OP pesticides. This reaction leads to the elimination of the leaving group, X. Figure 2.19 shows the reaction mechanism for OP pesticides in general.
N'.. /
.. N/ '---.
+
X
.. / N '-.,,_
Scheme 2 Mechanism of interaction of DQA with the OP pesticides in general.
75 The feasibility of the interaction of DQA is determined by the stability of the leaving group present in the OP pesticide. A better leaving group makes the reaction faster in comparison to a pesticide containing a poor leaving group. The leaving group which comes off more easily, the more stable it is as a free entity. This is usually inverse to its basicity, and the best leaving groups are the weakest bases. 14
If the structures of the individual four OP pesticides are taken into consideration, it is seen that ethion contains two electron deficient phosphorus atoms in its structure where nucleophilic attack of the sensor can take place. This explains why ethion binds with the sensor more strongly (and hence posseses the highest binding constant) compared to the other OP pesticides which contain a single electrophilic phosphorus atom. The non-interactive behavior of fenthion can also be explained by the stability of the leaving group. Both fenthion and parathion contain an aromatic system in their structures. However, as seen before, parathion interacts with DQA producing visible color changes, whereas fenthion is incapable of doing so. In the case of parathion interaction, the leaving group (2) is highly stable because of the presence of the electron withdrawing nitro group stabilizing the negative charge on the oxygen atom. However, for fenthion, the leaving group is (1) where both -SCH3 and -CH3 are electron donating groups, which increases the negative charge on the anion, making it very unstable resulting in the formation of a very unstable leaving group. This in part, explains the lack of interaction between fenthion and the sensor.
76 Figure 35 Structure of the leaving group in the case of parathion interaction .
.. N/ "'-.
.. / N '---.,
H,cs-0-0
Scheme 3 Mechanism of interaction of DQA with fenthion.
77 2.6 Conclusions
Sensors that can selectively detect and distinguish between various OP pesticides are in great demand for both environmental and agricultural applications.
We have successfully developed a new design of sensor that selectively binds to OP pesticides and upon binding, yields dual optical and electrochemical signal outputs based on the sensor-OP complex formed. Here, the sensor show three significant features: (1) the highly conjugated structure lead to strong visible colors, (2) the nitrogen atoms within the structure are nucleophilic due to presence of the lone pair of electrons, and hence permit binding to the electrophilic phosphorus atoms within the OP pesticides structure, and (3) the nitrogens being directly fused with the chromophore allows redox-active behavior that could be altered upon OP binding.
These three features were essential toward the development of selective sensor for OP pesticides.
Eventually, these sensors may be able to be used in a sensor array along with other sensors. This would improve the determination of the OP pesticides present because there would be more signals to observe which would reduce the risk of false positives.
78 2.7 References
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81 Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J .; Keith, T.; Al
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Revision B03; Gaussian, Inc.: Pittsburgh, PA, 2003.
82 CHAPTER3
WAVELENGTH-SPECIFIC SURFACE PLASMON RESONANCE SHIFTS IN GOLD NANOPARTICLES CAUSED BY ORGANOPHOSPHORUS PESTICIDES
3.1 Introduction
3.1.1 Advantages of using Gold Nanoparticles as Sensors toward Detection of Organophosphorus Pesticides
Metal nanoparticles are currently of great interest due to their applications in
various fields of science, namely, chemical catalysts, adsorbents, biological stains,
1 5 and elements of novel nanometer optical, electronic, and magnetic devices. · Lately,
these metal nanoparticles are emerging as important colorimetric reporters due to
their high extinction coefficients, which are several orders of magnitude larger
6 7 compared to organic dyes • and because the transition of the nanoparticles from
dispersion to aggregation exhibits a distinct change in color.7 Application of Gold
nanoparticles is worth-mentioning in this respect. This is because of the characteristic
plasmon absorption bands of gold nanoparticles which depend on their size and
9 10 shape. • The wide variation of the optical properties of gold nanoparticles with
particle size, particle-particle distance, and the dielectric properties of the surrounding
media due to surface plasmon resonance enables the construction of simple, yet
9 12 sensitive colorimetric sensors for various analytes. · The color change upon
aggregation is due to the coupling of the plasmon absorbances as a result of their
83 proximity to each other. By taking advantage of the mechanism of color transformation induced by the presence of analytes, gold nanoparticles have been
14 18 7 19 20 2 1 used for the detection of DNA, - several metal ions, • • and antibodies.
Recently, Organophosphorus based pesticide sensors based on nanoparticles
22 26 are being designed and reported by various groups. - The first work in this context was done by Pavlov et al. where Au NP plasmon resonance was utilized for optical determination of OP pesticides.22 On the other hand, Liu and Lin23 have reported an electrochemical OP pesticide sensor whose working principle is based on the affinity
24 25 of the analyte for ZrO2 nanoparticles. Blanchard et al. • have reported the synthesis of simple Au NP sensors functionalized to form a zirconium-phosphate (ZP) terminated surface for detection of OP pesticides. Zr4+ have a high affinity to bind
23 27 with OP pesticides, • whereas Au NPs served as optical reporting reagents, where the· plasmon resonance band of the Au NP is sensitive to the condition of the Zr-P linkage to its surface. However, the above mentioned nanoparticle based sensors suffered from several limitations. Firstly, the electrochemical method of detection reported by Liu and Lin23 is applicable only for nitroaromatic electroactive OP
4 25 pesticides. On the other hand, the sensing platform developed by Blanchard et al.2 • although simple and inexpensive, however the work presented is sensitive to the broad family of phosphate and phosphonate compounds, and hence suffers fromlack of selectivity. Also, no there has been no reports on investigation of the interaction of gold surface alone without funtionalization when it comes in contact with the OP pesticides.
84 The stability of a metal nanoparticle in solution results from the potential barrier that develops as a result of the competition between Van der Waals attraction and Coulomb repulsion between two nanoparticles.30 However, the stability against aggregation is largely a kinetic effect due to the large Coulombic repulsive barrier resulting from the localization of charged stabilizer capping the nanoparticle, making the effective charge on the nanoparticle zero. Various reports show that the addition of a neutral species to the stable nanoparticle solution can result in the destabilization
30 31 of the nanoparticle. • Addition of a neutral analyte which has high affinity to bind to the gold surface will replace the capping ligand which reduces the Coulombic barrier of repulsion between the nanoparticles, bringing them close together resulting in aggregation of the particles. In our work, we have utilized the aggregation-induced color changes of Au nanoparticles in aqueous solutions as an optical sensor toward the detection of OP pesticides. Gold nanoparticles of three differentsizes capped with
28 29 citrate were synthesized. • Citrate is a negatively charged weak capping ligand. On the other hand, gold is known to have a high affinity with sulfur atoms because of their soft character. Thus, when the thion pesticides containing sulfur atoms come in contact with the gold nanoparticles, the sulfur atoms will replace the negatively charged citrate ligand and bind to the gold surface releasing the capped citrate into the solution. This leads to reduction in the Coulombic repulsion barrier between two gold nanoparticles, which brings them in close proximity to each other, resulting in aggregation of Au nanoparticles accompanied by SPR shifts and visible color changes which can be readily detected by the naked eye.
85 Synthesis of aqueous suspensions of Au NPs involves the chemical reduction of HAuC14 solution by tri-sodium citrate to form gold crystals, which grow to form
13 gold nanoparticles by the process of Ostwald ripening. Varying the temperature and the ratio of HAuC14 and citrate used, three different sizes of Au NPs were obtained.
The interaction of these synthesized Au NPs was then investigated with three OP compounds, namely, ethion, malathion, and fenthion. Figure 36 shows the detection scheme of OP pesticides using the citrate capped gold nanoparticles.
citrate - Reduction -- -Citrate stabilized Au 3+ nanoparticles Au atoms 0 0 in dispersed form •• r• Au • OP e Au e e • • . e Au • • • I• Au • . . • •• An. t, ✓ . • • • • • ••• Au ... • An• e e • aggregation • ••
Figure 36 Detection scheme for OP pesticides with gold nanoparticles capped with citrate. Au NPs are stabilized with citrate which is a negatively charged weak capping ligand. Upon introduction of OP (small green circles) into the solution, nanoparticle aggregation is induced, which is manifested as a visible color change in the solution.
86 The novel method of sensing developed here has overcome several drawbacks
over the conventional methods of OP pesticides detection and offer three essential
features. (i) Firstly, the method is very simple and inexpensive since the synthesized
Au NPs do not need to through the tedious processes of functionalizing the gold
surface. Gold nanoparticles capped with citrate are very simple to synthesize, and also
possess high reproducibility and monodispersity. (ii) Secondly, the method was found
to be very selective toward specific pesticides, different optical signals were obtained
with each of the three OP compounds. (iii) Finally, the experiments involve water as
the solvent medium, thus ensuring proper functioning of the sensors in aqueous
environment. Figure 3 7 shows the structures of the OP compounds investigated in
this work.
/'----0 /--- /-- 0 � � �O-P-S� S-P-0 0 I oI �o �
Ethion Malathion
H3CS O - -OCH3 ,
H3CO H C ◊�3 Fenthion
Figure 37 Structures of the investigated Organophosphorus Pesticides.
87 3.2 Experimental Section
3.2.1 Materials
Sodium citrate, sodium borohydride, HAuC14.3H20, ethion, malathion, and fenthion were purchased from Aldrich chemicals and were used with no further purification. Deionized Milli-Q water at a pH of 7 was used to prepare all the aqueous solutions. Glassware were cleaned with acetone and dried in an oven before use.
3 .2.2 Transmission Electron Microscopy (TEM)
Gold nanoparticles were imaged usmg a JEOL transmission electron microscope (TEM), Model JEM-1230, Voltage 80 kV. Samples were prepared for electron microscopy by evaporating 1µ1 of the nanoparticle solution on formvar coated copper grids.
3.2.3 UV-visible Absorption Spectroscopy
A Varian Cary 50 UV-visible absorbance spectrophotometer was used to measure absorbance spectra. Quartz cuvettes with a 1 cm path length were used for all measurements. For a typical titration experiment, 1 µL aliquots of the OP
88 6 6 6 pesticides (ethion: lxl0- M, malathion: l.2x10- M, and fenthion: 1.4x10- M) were titrated to a 3 mL solution of the Au nanoparticle solution and SPR shifts were monitored until no change occurred. All titrations were performed at room temperature.
3.2.4 Synthesis of 4 nm Au Nanoparticles
Gold nanoparticles less than 4 nm were synthesized following an established procedure.28 Briefly, in a clean Erlenmeyer flask, 18.5 mL of deionized milli-Q
2 water, 0.5 mL of a l.0xl0- M aqueous HAuC14.3H2O solution, and 0.5 ml of a l.0xl0-2 M aqueous sodium citrate solution were placed. The resulting yellow colored solution was stirred for 2 min, and then 0.5 mL of a l.0xl0-2 M freshly prepared aqueous NaB� solution was added to it. Stirring was stopped as soon as NaB� was added to the solution resulting in a brownish red solution. This reaction resulted in the production of approximately 4 nm Au particles which was confirmed by transmission electron microscopy (TEM) as shown in Figure 38. The plasmon resonance band of these nanoparticles showed a maximum at 520 nm.
89 0.8
g 0.6 •0 � 0.4 I
0.2
0---�--�-�--�--� 300 400 500 60 700 800 Wavelength(nm)
Figure 38 (A) TEM images of 4 nm gold nanoparticles (B) UV-visible absorbance spectrum for 4 nm gold nanoparticles (inset showing the color of the 4 nm Au NP solution).
3.2.5 Synthesis of 13 and 30 nm Au Nanoparticles
Gold nanoparticles with average diameter 13 and 30 nm were synthesized
29 6 following a modified of published procedure. Briefly, 95 mL of l.21x10- M
° HAuC14.3H20 solution was heated at 75 C. While stirring this solution vigorously, 5 mL of 3 .4x 10-4 M sodium citrate was added. After about a minute a faint pink colored solution was obtained, which gradually darkened over a period of 5 mm. The resulting color of the solution was wme red. 30 nm gold nanoparticles were synthesized using the same procedure except the solution was heated at 85°C. TEM images confirmed that the resulting gold nanoparticles had average diameters of 13 and 30 nm respectively. The plasmon resonance band showed a maximum at 520 nm and 525 nm for13 and 30 nm Au NPs, respectively.
90 • • 0.6 ■
0.3 •' • 0 300 400 500 600 700 800 • Wavelength (nm)
Figure 39 (A) TEM images of 13 run gold nanoparticles (B) UV-visible absorbance spectrum for 13 run gold nanoparticles (inset showing the color of the 13 run Au NP solution).
• • ., • ., • 0.8 ' 0.6 0.4
0.2
' 0 • 300 400 500 600 700 800 ' Wavelength (nm)
Figure 40 (A) TEM images of 30 run gold nanoparticles (B) UV-visible absorbance spectrum for 30 run gold nanoparticles (inset showing the color of the 30 run Au NP solution).
91 3.3 Results and Discussion
The interaction of three different sizes of Au nanoparticles with the OP pesticides ethion, malathion, and fenthion was investigated. Citrate is a negatively charged capping ligand. On the other hand, gold is known to have a high affinity to bond with sulfur atoms because of their soft characters. Thus, when the thion pesticides containing several sulfur atoms come in contact with the gold nanoparticles, the sulfur atoms bind to the gold surface releasing the capped citrate into the solution. This leads into aggregation of Au nanoparticles because of the reduction in Coulombic repulsion between nanoparticles, resulting in visible color
3 9 changes accompanied by SPR shifts. -
3.3.1 Interaction of the 4 nm Gold Nanoparticles with Ethion, Malathion, and Fenthion
The interaction of each of the OP pesticides with 4 nm sized Au nanoparticles was investigated. 3 mL of the 4 nm Au NP solution was individually titrated with ethion, malathion, and fenthion, and the changes in the Au NP SPR were monitored by UV-visible absorbance spectroscopy. As shown in Figure 41, the addition of ethion in 1 µL (lxl0-6 M) increments to the 4 nm Au NP solution resulted in the decrease in the intensity of the 520 nm SPR peak, accompanied by the formationof a new peak at 790 nm. Increase in ethion concentration resulted in broadening of the
790 nm peak accompanied by red shifts. The color of the final solution was purple
92 indicating particle aggregation. Transmission electron micrograph of the Au nanoparticles after their interaction with ethion showed aggregation.
520 nm
0.8
0.6 .a .a 0.4
0.2
0 +------,------r------,----.,------r------, 300 400 500 600 700 800 900 Wavelength (nm)
0.6 • • • 0.5 • � • • 0.4 •
c( 0.3 • c( • 0.2
0.1
0 2 4 6 8 10 12
[Ethlon]*10-6 M
Figure 41 (A) UV-visible absorbance spectra of 4 nm Au nanoparticles in the presence of increasing concentrations of ethion. From top to bottom, [ethion] = 1, 2.1, 3.2, 4.2, 5.3, 6.3, 7.4, 8.5, 9.5, and 10.6 µM, respectively. (B) Plot of [Ethion] vs. change in SPR at 520 nm.
93 Titration of malathion in 1 µL (l.2xl0·6 M) increments with the 4 nm Au NP solution produced similar results as with ethion. However, in this case, as shown in figure 42, addition of malathion resulted in a decrease in the intensity of the 520 nm
Au NP SPR peak, accompanied by formation of a new peak at 770 nm. Increase in malathion concentration resulted in broadening of the 770 nm peak accompanied by red shifts. The color of the final solution was bluish black indicating particle aggregation. Transmission electron micrograph of the Au nanoparticles after their interaction with malathion showed aggregation.
520 nm
0.8
� 0.6 cuC .0 0 Cl) � 0.4
0.2
0-+-----�---�---�---�---�---� 300 400 50 600 70 800 90 Wavelength(nm)
Figure 42 (A) UV-visible absorbance spectra of 4 nm Au nanoparticles in the presence of increasing concentrations of malathion. From top to bottom, [malathion] = 1.2, 2.4, 3.6, 4.8, 5.9, 7.1, 8.3, 9.5, 10.7, and 11.9 µM, respectively. (B) A plot of [Malathion] vs. change in SPR at 520 nm.
94 Figure 42-Continued
0.7
� 0.6 • • • 0.5 • ,q; 0.4 • 0 • 0.3 • • 0.2 • 0.1 • • 0 0 2 4 6 8 10 12 14 [Malathion]*10"6M
Titration of fenthion in 1 µL (l.4x10-6 M) increments with the 4 nm Au NP solution produced exactly similar results as obtained with malathion. In this case, as shown in figure 43, addition of fenthion resulted in a decrease in the intensity of the
520 nm Au NP SPR peak, accompanied by formation of a new peak at 770 nm.
Increase in fenthion concentration resulted in broadening of the 770 nm peak accompanied by red shifts. The color of the final solution was bluish black indicating particle aggregation. Transmission electron micrograph of the Au nanoparticles after their interaction with fenthion showed aggregation.
95 1
� 520nm 0.8
0.6
<( 0.4
0.2
0+----�---�---�--�---�--� 300 40 500 600 70 800 9 Wavelength(nm)
0.7 • • 0.6 � • 0.5 • • <( 0.4 I • <( 0.3 •
0.2 •
0.1
0 0 2 4 6 8 10 12 [Fenthion]*10"6 M
Figure 43 (A) UV-visible absorbance spectra of 4 nm Au nanoparticles in the presence of increasing concentrations of fenthion.From top to bottom, [ fenth ion] = 1.4, 2.8, 4.3, 5.7, 7.1, 8.5, 9.9, 11.36, 12.8, and 14.2 µM, respectively. (B) Plot of [Fenthion] versus change in SPR at 520nm.
96 Figure 44 shows the aggregation of the 4 nm Au NPs after titration with ethion, malathion, and fenthion, and figure 45 shows the color changes in solution of
4 nm Au NPs after addition of the OP pesticides.
Figure 44 TEM image of (A) 4 nm Au NPs in the absence of any OP pesticides, and (B), (C), and (D) after titration with ethion, malathion, and fenthion respectively, indicating particle aggregation.
97 with with ,with J nm Au " ethion malathion fenthlon.
Figure 45 Color changes observed when OP pesticides ethion, malathion, and fenthion were added to the 4 nm Au nanoparticles.
3.3.2 Interaction of the 13 nm Gold Nanoparticles with Ethion, Malathion, and Fenthion
The interaction of three OP pesticides with 13 nm Au nanoparticles was investigated in a similar fashion by monitoring the changes in the Au NP SPR using
UV-visible absorbance spectroscopy. 3 mL of the 13 nm Au NP solution was individually titrated with ethion, malathion, and fenthion. However, 13 nm Au nanoparticles was found to be very selective in terms of binding with ethion, malathion, and fenthion, producing different colors of solutions with different Amax which could also be differentiated visually by naked eye.
6 As shown in figure 46 A, increase of ethion concentration in lµL (lxl0- M) increments to a solution of 13 nm Au NPs resulted in a decrease of the 520 nm resonance absorption band. Furthermore, a new broad peak appears at 700 nm and is accompanied by red shiftsas more ethion is introduced into the solution. The color of
98 the final solution was bluish black. No precipitation or cloudiness was observed. A plot of the red-shift in the visible extinction band maximum of the gold nanoparticle concentration versus [Ethion] reveals a linear relationship (Figure 46 B), indicating that this system can be used to quantitatively detect ethion in an aqueous medium.
Transmission electron microscopy clearly confirmed that particle aggregation had occurred upon ethion addition.
99 520 run
0.5
0+---�--�-�-�--�-�--�-�-�-� 300 350 400 450 500 550 60 650 700 750 800
Wavelength(nm)
0.16 • � • • 0.12 • • • c( � 0.08 • c( •
0.04 • • 0 0 2 4 6 8 10 12 14 [Eth ion]* 1 o·6 M
Figure 46 (A) Changes in the SPR peak of 13 runAu nanoparticles titrated with ethion. From top to bottom, [ethion] = 0, 1, 2.1, 3.2, 4.2, 5.3, 6.3, 7.4, 8.5, 9.5, and 10.6 µM, respectively. (B) Plot of [Ethion] vs. change in SPR at 520 run.
100 Addition of malathion to the 13 nm Au nanoparticles in 1 µL (l.2x10-6 M) increments did not show an immediate change in the Au SPR peak. Also, there was no change in the color of the solution at the end of titration, and TEM images did not show any significant changes with malathion addition. However, after 1 day, the 520 nm absorption peak decreased in intensity accompanied by formation of a new peak forms at 670 nm indicating particle aggregation. The color of the solution also changed to blue further confirming that the 13 nm Au nanoparticles were aggregated due to their interaction with malathion. We note that the 13 nm Au NPs in the absence of malathion were stable for days, at room temperature.
0.6
.Q 0.4 .Q
0.2
300 400 500 600 700 800 Wavelength (nm)
Figure 47 Changes in the SPR peak of 13 nm Au nanoparticles with increase in malathion concentrations. From top to bottom, [malathi�n] = 1.2, 2.4, 3.6, 4.8, 5.9, 7.1, 8.3, 9.5, 10.7, 11.9 µM, and after 1 day respectively.
101 6 Addition, of fenthion to a solution of 13 run Au NPs in 1 µL (l.4x10- M) increments resulted in a red-shift of the SPR from 520 run to 530 run, accompanied by formation of a new peak at 640 run. At the end of the titration, the color of the solution was purple. TEM images of the Au NPs after titration with fenthion showed particle aggregation.
0.8
520 run 0.6
C Ill .Cl 0 0.4
0.2
0 300 400 500 600 700 800
Wavelength (nm)
0.05 • 0.04 • • 0.03 c( • • c( 0.02 • • 0.01 • • 0 0 2 4 6 8 10 12 [Fenthlon]*10- M
Figure 48 Changes in the SPR peak of 13 run Au nanoparticles with increase in fenthion concentrations. From top to bottom, [fenthion] = 1.4, 2.8, 4.3, 5.7, 7.1, 8.5, 9.9, 11.36, 12.8, and 14.2 µM, respectively. (B) Plot of [Fenthion] versus change in SPR at 520 run.
102 Figure 49 shows the TEM images of the 13 nm Au NPs before and after titration with the different OP pesticides, and figure 3.15 shows the color changes in solution of 13 nm Au NPs after addition of the OP pesticides.
• - I .. � ., • •• •
50 ...
Figure 49 TEM image of (A) 13 nm Au NPs before, and (B), (C), and (D) after titration with ethion, malathion, and fenthion respectively. Particles have aggregated after titration.
103 Figure 50 Color changes observed with 13 nm Au nps after addition of the corresponding OP pesticides.
3.3.3 Interaction of the 30 nm Gold Nanoparticles with Ethion, Malathion, and Fenthion
The interaction of the three OP pesticides was investigated with 30 nm Au
nanoparticles in a similar fashion. 3 mL of the 30 nm Au NP solution was
individually titrated with ethion, malathion, and fenthion. In each case no immediate
changes in the SPR peak or color of the 30 nm Au NP solution was observed. TEM
images of the 30 nm Au NPs taken after - 6 hours showed no indication of
aggregation. However, after 24 hours, the Au NP solutions left at room temperature
containing ethion and fenthion changed from wine red to purple with a decrease in intensity of the 520 nm SPR peak. No changes were observed with malathion.
104 525 nm
0.4
C m .0
.0 c(
0-+------300 400 500 600 700 800 Wavelength(nm)
Figure 51 UV-visible absorbance spectra of 30 nm Au nanoparticles with increasing concentrations of ethion. From top to bottom, [ethion] = 1, 2.1, 3.2, 4.2, 5.3, 6.3, 7.4, 8.5, 9.5, and 10.6 µM, respectively.
105 0.8
0.6 525 nm
0.4
0.2
0-+------�---�----�----�---� 300 400 500 600 700 80 Wavelength(nm)
Figure 52 UV-visible absorbance spectra of 30 nm Au nanoparticles with increasing concentrations of malathion. From top to bottom, [malathion] = 1.2, 2.4, 3.6, 4.8, 5.9, 7.1, 8.3, 9.5, 10.7, and 11.9 µM, respectively.
106 0.6
C Ill .0 i 0.4
0.2
300 400 500 60 70 80
Wavelength(nm)
Figure 53 UV-visible absorbance spectra of 30 nm Au nanoparticles with increasing concentrations of fenthion. From top to bottom, [fenthion] = 1.4, 2.8, 4.3, 5.7, 7.1, 8.5, 9.9, 11.36, 12.8, and 14.2 µM, respectively.
Figure 54 TEM images of the~ 30nm Au NPs before addition of OP. No changes were observed in the images afteraddition of OPs.
107 3.4 Summary
In summary, we have demonstrated a new design of nanosensors which can distinguish between the organophosphorus pesticides ethion, malathion, and fenthion based on differentsizes of gold nanoparticles. The results are summarized in Table 5.
Au NP SPR shifts SPR shifts SPR shifts Detection
size with ethion with with f enthion limit
malathion
4nm 775nm 790nm 790nm 10"() M
13nm 700nm 520nm ( no 525nm 10"6 M changes (shoulder like immediately) peak forms 670 nm (after ~640nm) 1 day) 30nm No changes No changes No changes NIA immediately even after immediately several days
Table 5 Summary of results forthe interaction of the Au NPs of three different diameters with the OP pesticides.
Thus, as it can be seen, the 13 nm gold nanoparticles are very selective in binding with OP pesticides resulting in different Amax with each of them, whereas the
4 nm particles although is sensitive toward OP addition, however it lacks from selectivity; Figure 55 illustrates this data.
108 -8 �
B o.6
0 Ill � 0.4 -4nmAuNP -Bhion 0.2 -Mlthion -Fnthion
0+------�----�------� 300 400 500 600 700 800 Wavelength (nm)
0.8
� 0.6
0.4
-OM 0.2 -Bhion -Mllathion -Fenthion
0 300 400 500 600 700 800 Wavlength (nm)
Figure 55 (A) SPR shifts in 4 nm Au NPs caused by ethion, malathion, and fenthion. (B) SPR shifts in 13 nm Au NPs caused by ethion, malathion, and fenthion.
109 The observation that the 4 run and 13 runAu particles are more sensitive to OP pesticides in comparison to 30 run particles can be understood from simple surface
2 area arguments. 4 run nanoparticles have a surface area of ~50 run , whereas 13 nm
and 30 nm particles have surface areas of ~531 run2 and ~2820 run2 respectively.
Hence fewer molecules of OP pesticides are required to aggregate the smaller 4 run
and 13 nm nanoparticles. However, 4 run Au NPs are so small that it starts to show
aggregation instantaneously with addition of all the three OP pesticides that have
been investigated here. On the contrary, the surface area of 30 nm Au NPs is so large
that it requires more than a day to aggregate. But 13 run Au NPs were found to be
very selective in interaction with the OP pesticides producing different color changes
with different Aax•
The selectivity m interaction can also be accounted from the point of
consideration of the individual OP pesticide structures. Ethion possesses four sulfur
atoms in its structure, and hence can bind with more than one Au nanoparticle
simultaneously at a time bringing the nanoparticles closer which leads to
instantaneous aggregation with both 4 run and 13 run Au NP. Fenthion possesses two
sulfur atoms binding with two Au nanoparticle and bringing them closer enough to
cause aggregation. Due to smaller surface area in 4 run Au NPs, the aggregation with
fenthionoccurs instantaneously, but in the case of 13 run Au NPs, interaction is much
slower, taking more time (one day to develop a peak at longer wavelengths at 640
run), although the change in color of the solution was evident instantaneously with
addition of fenthion to 13 run Au NP. On the other hand, the position of sulfur atoms
in malathion is such that it does not enable to bind with more than one Au
110 nanoparticle simultaneously at a time because of steric hindrance, and hence the interaction of malathion with 13 nm Au NP was found to be very slow, visible changes not being observed beforea day. In order to confirm that Au-S interaction is taking place, titration experiments were carried out with paraoxon, containing no sulfur atom in its structure. It was observed that none of the three sizes of Au NPs were able to produce changes in color and UV-visible absorbance spectrum with paraoxon indicating that the presence of sulfuratoms causes the Au NPs to aggregate and produce visible color changes.
/o-�-o I "0
Figure 56 Structure of Paraoxon.
3.5 Conclusion
We have shown that Au NPs can detect and discriminate OP pesticides in an
aqueous environment. The detection is based on different color changes of Au
nanoparticles induced by OP pesticides producing wavelength-specific surface
plasmon resonance in the UV-visible spectra. 13 nm Au NPs were found to be very
selective toward binding OP pesticides producing specific color changes and Amax
with each of the pesticides. 4 nm Au NP was also found to be very sensitive toward
OP addition; however the very small surface area limits its selectivity toward OP
111 pesticides. On the other hand, nanoparticles of larger diameter ~ 30 run are limited to provide 'real-time' results, which restrict its application on the field. Thus, the effectiveness of the Au NPs toward OP detection is dependant on the particle size.
The system developed here can be effectively used to detect concentrations of OP pesticides in the ~ 10-6 M range and can be further exploited in future for selective detection of organophosphorus pesticides.
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