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Microbial Detection in Surface Waters

Jarod GregoryACCEND: Chemical Engineering B.S. & Environmental Engineering M.S.

Jon CannellChemical Engineering

Lilit Yeghiazarian, Ph.D.Environmental Engineering

Vasile Nistor, Ph.D.Biomedical Engineering

Creating a remotely-controlled mobile microbial biosensor

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Presentation Overview

• Introduction & Project Overview• Experimental Methods• Results• Future Work• Questions

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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

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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)

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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

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Introduction to Hydrogelspoly(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 Matter 2007, 3, 939.

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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.

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.

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Adsorption of Cationic Solute (slide 2 of 2)

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

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Functionalization of Hydrogel with E. Coli Antibodies via Glutaraldehyde (Slide 1)

NH2

NH2

NH2

E. Coli antibody from goat (representation to show presence of primary amines)

NH2

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.

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Functionalization of Hydrogel with E. Coli Antibodies via Glutaraldehyde (Slide 2)

NH2

NH2

NH2NH2

Glutaraldehyde cross-linking primary amines

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Hydrogel functionalized for e.

coli capture

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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

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Fluorescent Imaging ResultsFluorescent imaging was used to verify primary antibody attachment via the detection of the presence of Alexa-647 labeled secondary antibodies

E. Coli Primary Antibody Exp.

Campylobacter Primary Antibody Exp.

Cryptosporidium Primary Antibody Exp.

Control Sample Control Sample Control Sample

Acriflavine YES YES YES YES YES YES

PrimaryAntibody

NO Goat NO Mouse NO Goat

Secondary Antibody

Anti-Goat

Anti-Goat Anti-Mouse

Anti-Mouse

Anti-Goat Anti-Goat

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E. Coli Antibody Attachment Results (slide 1 of 2)

Fluorescent images of samples excited by 488 nm single photon laser

SampleControl

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Fluorescent images of both samples excited by 640 nm laser*Images have 70% enhanced brightness

Control

E. Coli Antibody Attachment Results (slide 2 of 2)

Sample

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Cryptosporidium Antibody Attachment Results (slide 1 of 2)

Fluorescent images of samples excited by 488 nm single photon laser

Control Sample

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Fluorescent images of both samples excited by 640 nm laser*Images have 70% enhanced brightness

Cryptosporidium Antibody Attachment Results (slide 2 of 2)

Control Sample

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Campylobacter Jejuni Antibody Attachment Results (slide 1 of 2)

Fluorescent images of samples excited by 488 nm single photon laser

SampleControl

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Fluorescent images of both samples excited by 640 nm laser*Images have 70% enhanced brightness

Campylobacter Jejuni Antibody Attachment Results (slide 2 of 2)

SampleControl

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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

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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

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Questions?