1. Microbial Detection in Surface Waters Jarod Gregory ACCEND: Chemical Engineering B.S. & Environmental Engineering M.S. Jon Cannell Chemical Engineering Lilit Yeghiazarian, Ph.D. Environmental Engineering Vasile Nistor, Ph.D. Biomedical Engineering 1 Creating a remotely-controlled mobile microbial biosensor

2. Presentation Overview Introduction & Project Overview Experimental Methods Results Future Work Questions 2

3. Microbe Candidates Escherichia coli –According to the EPA, approximately 93,000 river and stream miles contain elevated bacterial levels Cryptosporidium –548 outbreaks from 1948-1994 –Spore-forming protozoa –Tolerant to chlorine disinfection Campylobacter Jejuni –Inflammatory, exudative enteritus –Can cause Guillain-Barre syndrome –Common to many bird species 3 Campylobacter Jejuni – en.wikipedia.org/wiki/campylobactor

4. Project Overview The long-term goal of this project is to create an autonomous hydrogel biosensor capable of detecting microbials in surface waters and transmitting contamination information in real time or near-real time This would be a qualitative leap in detection/tracking capabilities, as the current process requires physical samples taken to a lab (24-hour turnaround) 4

5. Project Overview Phase I: Proof-of-principle of peristaltic motion in free-floating hydrogels Phase II: Functionalize the hydrogels with the capability to capture E. coli and other microbials Phase III: Internalize propulsion mechanism Phase IV: Transmission of microbial detection data 5

6. Introduction to Hydrogels poly(N-isopropyl) acrylamide (PNIPAM) hydrogels are synthetic gels that consist almost entirely of absorbed water, giving them flexibility similar to natural tissue –PNIPAM hydrogels undergo a dramatic volume phase transition at a critical temperature (LCST) of approximately 33 o C [1] 6 Our ‘fast’ hydrogels use a synthetic layered silicate called Laponite as a cross-linker and are synthesized above the LCST in order to increase strength and improve absorption dynamics [1] L. Yeghiazarian, H. Arora, V. Nistor, C. Montemagno, U. Wiesner, Soft Matter 2007, 3, 939.

7. The Laponite cross-linker that is part of the hydrogel’s structure not only strengthens the hydrogel, but gives it the ability to adsorb positively- charged solutes out of solution. Adsorption of Cationic Solute (slide 1 of 2) The ability to effectively adsorb and retain positively-charged molecules gives hydrogels a wide platform for conjugation opportunities and is the basis for our REU project. [2] P. C. Thomas, B. H. Cipriano, S. R. Raghavan, Soft Matter 2011, 7, 8192–8197. 7 Image of a cross-section of a cylindrical PNIPAM hydrogel that has adsorbed IR-820 dye being excited with an 820 nm laser. This image shows the nature of the IR -820’s adsorption, which is localized along the surface of the hydrogel.

8. Adsorption of Cationic Solute (slide 2 of 2) 8 1. Allow the hydrogel to immerse in acriflavine/water solution and adsorb the cationic solute 2. Hydrogel w/ portion that has adsorbed the acriflavinium chloride HYDROGEL

9. Functionalization of Hydrogel with E. Coli Antibodies via Glutaraldehyde (Slide 1) 9 NH 2 E. Coli antibody from goat (representation to show presence of primary amines) NH 2 Hydrogel w/ exposed primary amines from acriflavine adsorption Glutaraldehyde is the most popular homobiofunctional cross-linker, which joins two molecules (usually antibody  enzyme) via a number of mechanisms of reactivity with primary amines.

10. Functionalization of Hydrogel with E. Coli Antibodies via Glutaraldehyde (Slide 2) 10 NH 2 Glutaraldehyde cross-linking primary amines 1 Hydrogel functionalized for e. coli capture 2

11. Verifying E. Coli Antibody Attachment 11 Donkey anti-Goat (DaG) anitbody is used as a fluorescent ‘stain’ Will only attach to a goat antibody Labeled with Alexa 647, which can be imaged using fluorescent microscopy Alexa 647 label

12. Fluorescent Imaging Results Fluorescent imaging was used to verify primary antibody attachment via the detection of the presence of Alexa-647 labeled secondary antibodies 12 E. Coli Primary Antibody Exp. Campylobacter Primary Antibody Exp. Cryptosporidium Primary Antibody Exp. ControlSampleControlSampleControlSample AcriflavineYES Primary Antibody NOGoatNOMouseNOGoat Secondary Antibody Anti- Goat Anti- Mouse Anti-Goat

13. E. Coli Antibody Attachment Results (slide 1 of 2) 13 Fluorescent images of samples excited by 488 nm single photon laser SampleControl

14. Fluorescent images of both samples excited by 640 nm laser *Images have 70% enhanced brightness Control 14 E. Coli Antibody Attachment Results (slide 2 of 2) Sample

15. Cryptosporidium Antibody Attachment Results (slide 1 of 2) 15 Fluorescent images of samples excited by 488 nm single photon laser ControlSample

16. Fluorescent images of both samples excited by 640 nm laser *Images have 70% enhanced brightness 16 Cryptosporidium Antibody Attachment Results (slide 2 of 2) ControlSample

17. Campylobacter Jejuni Antibody Attachment Results (slide 1 of 2) 17 Fluorescent images of samples excited by 488 nm single photon laser SampleControl

18. Fluorescent images of both samples excited by 640 nm laser *Images have 70% enhanced brightness 18 Campylobacter Jejuni Antibody Attachment Results (slide 2 of 2) SampleControl

19. Future Work Repeat the experiments for campylobacter jenuni primary antibody conjugation Prove that the functionalized hydrogel can capture heat-killed E. coli cells Internalize a mobility mechanism and make the hydrogel capable of transmitting contamination data to a central location 19

20. Acknowledgements Professors Yeghiazarian and Nistor National Science Foundation “EAGER: Monitoring Our Nation’s Waters – Towards a Swimming Biosensor to Dynamically Map Microbial Contamination” Grant National Science Foundation Research Experience for Undergraduates Program 20

21. Questions? 21