Flourescence-Based Detection of Pesticides via Conjugated Polymer Nanoparticles

The use of synthetic pesticides has played a large role in increasing crop yields throughout the world, but their adverse effects on humans and non-target animals is of major concern due to their toxicity and persistence in the environment. Some of the more persistent examples are organochlorine pesticides, particularly dichlorodiphenyltrichloroethane (DDT) and its metabolites. Reported herein is the development of a detection scheme using organic nanoparticles for the fluorescence detection of a range of pesticides. The nanoparticles were fabricated from a synthetic conjugated fluorescent polymer, and fluorescence experiments were performed using both nanoparticle solution and polymer thin films. The large extinction coefficients exhibited by conjugated fluorescent polymers (also referred to as conjugated amplifying polymers), such as the one discussed herein, make them useful for chemical detection schemes. In order to maintain this strong fluorescence of the polymer in solution, it must be in an aggregated state, which allows for both intra-polymer and inter-polymer exciton transfer. To achieve this aggregated state in solution, the formation of polymer nanoparticles is used. These nanoparticles allow the polymer to be used for chemical detection of pesticides in solution via fluorescence enhancement. The 2,1,3-benzooxadiazole-alt-fluorene (PFBO) polymer nanoparticles discussed herein were fabricated using the reprecipitation method, which is the formation of spherical particles as a result of the hydrophobic collapse of the polymer in an aqueous solution, and average particle size was confirmed using dynamic light scattering. In solution, a limit of detection of 4.5 ppm was achieved for DDT in the presence of the PFBO nanoparticles.


FIGURE PAGE
Chapter 1:

INTRODUCTION
The widespread use of pesticides has been highly effective in increasing the harvested yields of many crops worldwide through eliminating the threat of common pests, but their use has also been of concern due to their known and suspected toxicity to humans and other species and long term environmental persistence. 1 One class of pesticides that is of continuing concern is organochlorine pesticides (OCPs), the most common of which is dichlorodiphenyltrichloroethane (DDT), sold commercially as a mixture of the para, para-(compound 1, Chart 1) and ortho, para-(compound 4) and dichlorodiphenyldichloroethylene (DDE, compound 3) are some of the primary metabolites of DDT, also with known toxicities. 3 Other pesticide classes of interest include: (a) aliphatic organochlorines 5 and 6; (b) carbamate pesticides 7 and 8, which are less environmentally persistent but still pose acute health risks; 4 and (b) synthetic pyrethroids 9 and 10, which are less acutely toxic and less environmentally persistent, and have been increasing in usage in recent years. 5 Techniques for the detection of organic pesticides generally rely on chromatography followed by mass spectrometry. 6 These methods offer good sensitivity and resolving power, but suffer from the high cost of operation and tedious and timeconsuming sample preparations, 7 which limits the ability to conduct high throughput assays. Newer techniques for pesticide detection include molecularly imprinted polymer systems, 8 nanoparticle-based immunoassays, 9 and gold nanoparticle-based Raman spectroscopy. 10 A variety of fluorescence-based methods for pesticide detection have also been reported, 11 although in many cases these methods require derivatization steps, 12 chromatographic purification, 13 and/or are substantially limited in terms of the range of pesticides that can be detected. 14 One method of detection that has shown a lot of promise in the detection of multiple classes of analytes with extremely high sensitivity and selectivity is the use of conjugated fluorescent polymer sensors. 15 Typically, detection efficiencies are optimal in polymer aggregates such as thin films 16 or conjugated nanoparticles, 17 which enable inter-polymer as well as intra-polymer exciton migration. 18 Formation of conjugated polymer-derived nanoparticles can occur through a variety of methods, 19 including reprecipitation, 20 in which the hydrophobic polymer collapses upon its introduction into aqueous solution, resulting in the formation of well-defined spherical nanoparticles. λ max emission: polymer = 507 nm; particles = 534 nm). 23 The concentration of 11 was varied (see ESI for more details), and optimal fluorescence responses were obtained with a 1.25 x 10 -3 mg/mL polymer solution.
Results of the fluorescence modification experiments are shown in Table 1, and key trends are discussed in further detail below.     Literature precedent by Swager and co-workers demonstrated that fluorescent polymer thin films underwent substantial fluorescence enhancements as a result of analyte-mediated reduction of the polymer chain, an effect that was easily reversed by introduction of iodine for re-oxidation. 26 Other examples of the susceptibility of conjugated polymer-derived nanoparticles to oxidation and reduction have also been reported. 27    An extension of this fluorescence-based detection to polymer 11-derived thin films was conducted by fabricating fluorescent thin films from the spin casting of a polymer 11 solution in chloroform onto glass slides. These films were briefly exposed to the vapor from a solution of DDT 1 in tetrahydrofuran. The measurable response of these films to DDT vapor ( Figure 6A) is remarkable considering the low vapor pressure of DDT, 29 and indicates high levels of sensitivity in these fluorescent polymer-derived detection systems. Moreover, control experiments indicated that the tetrahydrofuran itself had negligible effects on the photophysical properties of polymer 11 -derived thin films.
These fluorescence changes were also reversible with exposure of the thin film to iodine vapor, leading to a nearly complete return to the initial thin film fluorescence state (127% increase followed by 120% decrease, Figure 6).  than 'turn-off' fluorescence signal, which has the potential to lead to improved sensitivity in practical detection schemes; and (e) low limits of detection, which approach practical levels of concern in some cases. Efforts towards developing practical turn-on detection systems for aromatic pesticides based on this research are currently in progress in our research laboratory, and results of these and other investigations will be reported in due course.

ACKNOWLEDEMENTS
Funding for this research was provided by the University of Rhode Island Chemistry Department start-up funds.

Notes and References
Supporting Information

MATERIALS AND METHODS
All the starting materials, reagents, and solvents were purchased from Sigma Aldrich, Acros Organics, TCI chemicals, Alfa Aesar, or Fisher Scientific and were used as Dynamic light scattering experiments were run on a Malvern Zetasizer Nano ZS90, measuring particle size at 25°C and a 90° measurement angle, using Mark-Houwink parameters for the calculation of molecular weight.
Gel permeation chromatography (GPC) data were obtained using an Agilent Infinity  For the LOD, the limit of the blank was defined by the following equation: Where m is the mean of the blank integrations and SD is the standard deviation.
The LOB value was then inserted into the line equation as the Y-value, and the Xvalue was solved for, giving the LOD in mM.
For the LOQ, the limit of the blank was defined by the following equation: The LOB value was then inserted into the line equation as the Y-value, and the Xvalue was solved for, giving the LOQ in mM.

SUMMARY TABLES FOR THIN FILM EXPERIMENTS
Ratio of fluorescence in thin films with DDT and I 2 additions: Ratio is defined as the integrated fluorescence of the film under a given set of experimental conditions to the integrated fluorescence of the film before treatment with any analyte or reagent.  Research indicates that this gender gap may start as early as elementary school, with female students having a more negative attitude towards science than males starting as early as 4 th grade. 3,4 This gender gap is likely reinforced by the fact that high school science teachers spend significantly more time addressing the boys in the classroom, a fact that has been well-documented in the literature as recently as 2013. 5,6 This gender gap has a multitude of potential causes that have been investigated in the literature, including: (a) a lack of female scientist role models, 7,8 which contributes to childrens' perceptions that scientists are overwhelmingly white males; 9,10 (b) girls' self-perception that they lack aptitude and ability to succeed in STEM disciplines; 11 and (c) teachers', parents', and other authority figures' reinforcement of these stereotypical notions. 12,13 These phenomena affect children as young as 4 years old, 14 and continue to affect students' attitudes, perceptions, and experiences throughout their K-12 education, ultimately culminating in significant gender gaps in college students' choices of majors and careers. [15][16][17] Educators have attempted to address this gender gap through increasing girls' access to female role models, [18][19][20] and through conducting outreach activities specifically targeted towards female students. 21,22 A concurrent problem in STEM education is the lack of hands-on laboratory time in the formal middle school and high school curricula, which is attributable to a general decrease in funding for STEM education, 23,24 as well as an increased prevalence of standardized testing that de-emphasizes hands-on experimental training. 25 To address this issue, educators have conducted hands-on outreach workshops, [26][27][28] developed creative methods to increase the time devoted to hands-on learning, 29,30 and implemented innovative uses of technology to conduct virtual field trips 31 and virtual science experiments. 32,33 To simultaneously address both of these issues: the persistent gender gap in STEM disciplines and the lack of hands-on science education, we developed a full- Participants were responsible for arranging their own transportation to and from camp each day. In addition to the 11 major activities discussed below, students also participated in multiple swimming breaks throughout the week, watched selected science videos, and engaged in extensive interactions with invited speakers, camp volunteers, and the PI, Dr. Levine.

PARTICIPANT DEMOGRAPHICS
The 36 participants came from communities throughout the state of Rhode Island, with the largest contingent from Pawtucket (9/36 of the girls). The participants came from public schools (17), private schools (14), charter schools (1), and home schools (4). 25% of the girls were from non-white minority groups (9/36).

HANDS ON EXPERIMENTATION
As mentioned in the introduction, one goal of the camp was to educate the participants about the applicability of science in their everyday lives through hands-on experimentation. This hands-on experimentation has been shown to be crucial to encouraging general interest in and enthusiasm about STEM disciplines. 39 To that end, the camp schedule included 11 hands-on activities (Table 1). For each activity, the participants learned about the key scientific background, conducted the experiments, and discussed the results. Selected photographs of these activities are shown in Figure   1.   60 and in particular asked the participants to rate their responses to the questions shown in Table 2 on a scale of 1-5 (1 = strongly agree; 5 = strongly disagree). Asterisks next to the question numbers indicate those questions that had the most significant differences in responses pre-and post-camp.
The results of this survey are summarized in Table 2. A paired t-test conducted on this data gave a two-tailed P value less than 0.0001 for the cumulative survey scores,  The scale for the survey item response scores is 1-5, with 1 indicating "strongly agree" and 5 indicating "strongly disagree". c 28 of the 36 participants consented to participate in this study; the results reported herein are based only on the surveys of the 28 consenting participants.
(2) The questions with the greatest pre-camp to post-camp differential were, "Science can help me figure out how to spin/shoot/throw/hit the ball" (Question 7), and "Using scientific methods helps me decide what to buy in the store" (Question scientist discussion) can be run as an independent event, and is also likely to increase the participants' excitement for and exposure to science.
One unanswered question is whether the positive effects observed in the survey responses will persist long-term, with girls who have participated in this program maintaining their scientific enthusiasm over subsequent months and years. Future efforts will focus on conducting follow-up surveys of the program participants, to track their long-term interest in science, as well as their choice of college, college major, and future career. In future years, we will also administer more detailed surveys to elucidate the effects of each aspect of this program (experiments, field trips, and scientist interactions) on impacting girls' attitudes about science. This ongoing outreach activity at the University of Rhode Island is currently being funded by private and corporate donations.

III. You Clean that Oil Spill, Diaper Polymer:
Sodium polyacrylate is also used to clean oil spills. We will recreate a small oil spill and see how well the technique works. separate a complex mixture into its individual components. Paper chromatography has two phases: a stationary phase (the paper) and a mobile phase (the solvent). Depending on the characteristics of each pigment, they will either have a high affinity for the stationary phase (so they will not move as much up the paper) or they will have a high affinity for the mobile phase (they will move far up the paper). Because of this, we can manipulate the mobile phase as needed to ensure that each of the spots on the chromatogram (the chromatography paper after the pigments have been separated) are far enough apart so we can clearly differentiate between components. By comparing the chromatogram from an unknown sample to a series of known samples, we can identify what the unknown sample is.
1. Obtain 2-4 strips of chromatography paper (depending on how thick it is, you may be able to do 1-2 spots on each paper). Your instructors will tell you how many to use.
2. Using a ruler, draw using a pencil (not pen: ink is made of different compounds, so it too will be separated if used on chromatography paper) a line ~2 cm from the bottom of the strips of filter paper.
3. Label each piece of paper with the sample(s) of lipstick that will be on the paper.  contains water and a colored wax in the top chamber and a light at the base.
The wax that is used is paraffin wax, which is also used in candles. Wax has similar properties to oil, and as such the wax normally sits on top of the water because it is less dense (lighter in weight) to water, which is more dense (heavier weight). However, in Figure 1 (2), we see that the wax actually rests on the bottom when the lamp is off. This is due to an additive that the manufacturers mix with the wax that make it more dense (heavier) than water, so the wax sits at the bottom, near the lamp that is in the base.
When the lamp is turned on, the wax (being right on top of it) becomes heated and expands, making it less dense than water. As a result, it rises above the water (Figure 1, red line, (3)). Once the wax reaches the top of the lamp, the wax cools just enough (because it is not near the lamp anymore) to increase its density and it falls to the bottom of the lamp (Figure 1, blue line, (3)). This process repeats until the lamp is turned off.
Salts are formed when ions (one positively charged and the other negatively charged) come together to make a neutral compound. Table salt is sodium chloride, NaCl, with sodium being positively charged (Na+) and chloride being negatively charged (Cl-). When you buy salt, it is a solid (NaCl).
However, when you add salt to water, it dissolves. The reason for this is because water can "pull apart" the solid salt into its individual components (Na+ and Cl-) which are water soluble. Water contains a positive end and a negative end. Because opposites attract, the positive sodium associates with the negative end of the water, while the negative chloride associates with the positive end of water. These different associates make it water soluble. This association arises from ionic bonds, in which opposite charges attract one another. sphere. They only last a few seconds before they pop because the water evaporates (although this can be delayed with glycerin), or they make contact with an object. The bubbles tend to change color and/or show a rainbow pattern, and this occurs when light is reflecting off the front and back surface of the bubble, causing interference with one another (this is why the colors appear to "swirl" around the bubble surface). Bubbles are hollow spheres because this shape is best to enclose as much air as possible with as little bubble solution as possible. This is also due to the attractive forces between the molecules in the bubble; think of it like a bunch of friends holding hands and running around. It's easier to move in a circle instead of a square or rectangle or triangle, and in molecules this all relates to surface area.

Bubble Solution
4. Slide a straw over each pipe cleaner. We do this because the bubbles would soak the pipe cleaner, and we would not be able to create bubbles with it. 5. Start joining the four pyramids together by twisting the ends together, and continue until you form a cube.

TIE DYE
VII. Make your own Tie Dye Shirt: Learn the science behind how tie dyeing a shirt works. To tie dye a shirt, the shirt is first soaked in a solution of sodium carbonate. Sodium carbonate is a common chemical that you can find in your home as it is the main chemical found in laundry detergent and bubble bath solutions (although this is much more concentrated!). Cotton t-shirts contain mostly cellulose. When sodium carbonate, a weak base, is added, the pH is raised. As a result, the hydrogen that was bonded to the oxygen (seen in blue with green square) "leaves" and what results is a negatively charged oxygen (seen in blue with red square) and sodium bicarbonate (baking soda!). This negatively-charged oxygen is now an open bonding site for our dye. As we add different dyes to the shirt, these bonding sites "capture" the dye and thus the shirt goes from white (when it was regular cellulose) to whatever color you've chosen ( Figure S4). Part E: Washing the Shirt 1. This is very important: the first wash affixes the color. Be sure to do these steps exactly.
2. After 24 hours, unwrap the shirt and remove the rubber bands. Rinse the shirt in equal parts cold water and white vinegar until the shirt no longer feels soapy. When this happens, the pH of the shirt is neutral (pH=7).
3. The dye will not stain drains but will stain other fabric, so be careful when washing the shirt. Also be sure you're wearing gloves! Part A the rough surface causes nucleation of the carbon dioxide. In other words, the gas bubbles are able to collect in the ridges of the candy, so the many small bubbles we usually see quickly multiply until the pressure caused is released, and that's when we see the explosion of soda! So, the last question is why does this work better with Diet Coke than other sodas? It is believed that the reason is that Diet Coke uses different ingredients and is less sticky compared to other soda formulations, so it is much more effective at producing the carbon dioxide gas needed to get a larger soda explosion.

X. Classifying Fluids:
A fluid is a substance with no definite shape, and is easily deformed by outside pressure. Any liquid or gas is a fluid.
Viscosity is the property of a fluid that describes how easily it can flow. If a substance doesn't flow easily, it's said to be viscous. So we could call molasses viscous, when compared with water.
A Newtonian fluid is a fluid that has a constant viscosity. Water is a Newtonian fluid. These fluids behave as you'd expect them to. They're called Newtonian, because Isaac Newton found equations to correctly describe their behavior.
A Non-Newtonian fluid has a viscosity that changes under different conditions. These can get either more viscous or less viscous when pressure is applied.
to do the serious mixing. Keep adding the Borax solution to the glue mixture (don't stop mixing) until you get a perfect batch of Elmer's slime. 6. When you're finished playing with your Elmer's slime, seal it up in a zipper-lock bag for safekeeping. pH PAPER XI. Make Your Own pH Paper: Red cabbage contains a pigment molecule called flavin (an anthocyanin). This water-soluble pigment is also found in apple skin, plums, poppies, cornflowers, and grapes. Very acidic solutions will turn anthocyanin a red color. Neutral solutions will result in a purple color. Basic solutions appear in greenish-yellow. Therefore it is possible to determine the pH of a solution based on the color it turns the anthocyanin pigments in red cabbage 2. Place your 5 (or 6) pieces of filter paper on the bottom of your beaker.
Then ask your instructor to pour more solution into the beaker to cover the filter paper.
3. Notice that each of your test tubes is labeled with the name of a household chemical, and that one of these chemicals is on your