D etection in Surface Waters Jarod Gregory ACCEND Chemical Engineering BS amp Environmental Engineering MS Jon Cannell Chemical Engineering Lilit Yeghiazarian PhD Environmental Engineering ID: 202774
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Slide1
Microbial Detection in Surface Waters
Jarod GregoryACCEND: Chemical Engineering B.S. & Environmental Engineering M.S.Jon CannellChemical EngineeringLilit Yeghiazarian, Ph.D.Environmental EngineeringVasile Nistor, Ph.D.Biomedical Engineering
1
Creating a remotely-controlled mobile microbial biosensorSlide2
Presentation Overview
Introduction & Project OverviewExperimental MethodsResultsFuture WorkQuestions2Slide3
Microbe Candidates
Escherichia coliAccording to the EPA, approximately 93,000 river and stream miles contain elevated bacterial levelsCryptosporidium548 outbreaks from 1948-1994Spore-forming protozoaTolerant to chlorine disinfectionCampylobacter JejuniInflammatory, exudative enteritusCan cause Guillain-Barre syndromeCommon to many bird species3
Campylobacter
Jejuni – en.wikipedia.org/wiki/
campylobactorSlide4
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 timeThis would be a qualitative leap in detection/tracking capabilities, as the current process requires physical samples taken to a lab (24-hour turnaround) 4Slide5
Project Overview
Phase I: Proof-of-principle of peristaltic motion in free-floating hydrogelsPhase II: Functionalize the hydrogels with the capability to capture E. coli and other microbialsPhase III: Internalize propulsion mechanismPhase IV: Transmission of microbial detection data5Slide6
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 tissuePNIPAM hydrogels undergo a dramatic volume phase transition at a critical temperature (LCST) of approximately 33 oC [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. Slide7
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.
Slide8
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
HYDROGELSlide9
Functionalization of Hydrogel with E. Coli Antibodies via Glutaraldehyde
(Slide 1)9
NH
2
NH
2
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. Slide10
Functionalization of Hydrogel with E. Coli Antibodies via Glutaraldehyde
(Slide 2)10
NH
2
NH
2
NH
2
NH
2
Glutaraldehyde
cross-linking primary amines
1
Hydrogel functionalized for e. coli capture
2Slide11
Verifying E. Coli Antibody Attachment
11Donkey anti-Goat (DaG) anitbody is used as a fluorescent ‘stain’Will only attach to a goat antibodyLabeled with Alexa 647, which can be imaged using fluorescent microscopy
Alexa
647 labelSlide12
Fluorescent Imaging Results
Fluorescent imaging was used to verify primary antibody attachment via the detection of the presence of Alexa-647 labeled secondary antibodies12E. Coli Primary Antibody Exp.Campylobacter Primary Antibody Exp.
Cryptosporidium Primary Antibody Exp.
ControlSample
Control
Sample
Control
Sample
Acriflavine
YES
YES
YES
YES
YES
YES
Primary
Antibody
NO
Goat
NO
Mouse
NO
Goat
Secondary Antibody
Anti-Goat
Anti-Goat
Anti-Mouse
Anti-Mouse
Anti-Goat
Anti-GoatSlide13
E. Coli Antibody Attachment Results (slide 1 of 2)
13Fluorescent images of samples excited by 488 nm single photon laserSample
ControlSlide14
Fluorescent images of both samples excited by 640 nm laser
*Images have 70% enhanced brightnessControl14
E. Coli Antibody Attachment Results
(slide 2 of 2)
SampleSlide15
Cryptosporidium Antibody Attachment Results (slide 1 of 2)
15Fluorescent images of samples excited by 488 nm single photon laserControl
SampleSlide16
Fluorescent images of both samples excited by 640 nm laser
*Images have 70% enhanced brightness16
Cryptosporidium Antibody Attachment Results
(slide 2 of 2)
Control
SampleSlide17
Campylobacter Jejuni Antibody Attachment Results
(slide 1 of 2)17Fluorescent images of samples excited by 488 nm single photon laserSample
ControlSlide18
Fluorescent images of both samples excited by 640 nm laser
*Images have 70% enhanced brightness18
Campylobacter
Jejuni
Antibody Attachment Results
(slide 2 of 2)
Sample
ControlSlide19
Future Work
Repeat the experiments for campylobacter jenuni primary antibody conjugationProve that the functionalized hydrogel can capture heat-killed E. coli cellsInternalize a mobility mechanism and make the hydrogel capable of transmitting contamination data to a central location19Slide20
Acknowledgements
Professors Yeghiazarian and NistorNational Science Foundation “EAGER: Monitoring Our Nation’s Waters – Towards a Swimming Biosensor to Dynamically Map Microbial Contamination” GrantNational Science Foundation Research Experience for Undergraduates Program20Slide21
Questions?
21