Microbial D etection in Surface Waters

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

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

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 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)

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

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 oC [1] 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 Matter2007, 3, 939.

7. Adsorption of Cationic Solute (slide 1 of 2) 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. 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. 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 Matter2011, 7, 8192–8197.

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

9. Functionalization of Hydrogel with E. Coli Antibodies via Glutaraldehyde (Slide 1) 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. NH2 NH2 NH2 NH2 E. Coli antibody from goat (representation to show presence of primary amines)

10. Functionalization of Hydrogel with E. Coli Antibodies via Glutaraldehyde (Slide 2) 1 2 NH2 NH2 Hydrogel functionalized for e. coli capture NH2 NH2 Glutaraldehyde cross-linking primary amines

11. Verifying E. Coli Antibody Attachment • 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

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

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

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

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

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

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

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

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

21. Questions?