Technical Paper
Microbial Detection in Surface Waters
Submitted To
2013 Academic Year NSF AY-REU Program
Part of
NSF Type I STEP Grant
Sponsored By
The National Science Foundation
Grant ID No:
College of Engineering and Applied Sciences
University of Cincinnati
Cincinnati, Ohio
Prepared By:
Jarod Gregory, Pre-Junior, ACCEND Chemical Engineering B.S., Environmental Engineering M.S.
Jonathon Cannell, Pre-Junior, ACCEND Chemical Engineering B.S., M.B.A.
Reviewed By:
______________________________________________
Dr. Lilit Yeghiazarian
REU Project Mentor
Assistant Professor
School of Biomedical, Chemical, and Environmental Engineering
University of Cincinnati
September 13 – December 5 2013
0
Developing a Hydrogel-Based Microbial
Biosensor
By Jarod Gregorya, Jonathon Cannellb, Lilit Yeghiazarian*, and Vasile Nistor*
Keywords: Hydrogel, Biosensor, E. coli, Cryptosporidium, Acriflavine
*Corresponding Authors
Drs. Lilit Yeghiazarian and Vasile Nistor
School of Biomedical, Chemical, and Environmental Engineering
University of Cincinnati
2600 Clifton Ave., Cincinnati, OH 45221
a
Jarod Gregory
ACCEND – Chemical Engineering B.S., Environmental Engineering M.S., Class of 2015
School of Biomedical, Chemical, and Environmental Engineering
University of Cincinnati
2600 Clifton Ave., Cincinnati, OH 45221
b
Jonathon Cannell
ACCEND – Chemical Engineering B.S., M.B.A., Class of 2015
School of Biomedical, Chemical, and Environmental Engineering
University of Cincinnati
2600 Clifton Ave., Cincinnati, OH 45221
Abstract
Microbial contamination of surface waters is an important economical, health-related, and
engineering issue facing the U.S. and countries throughout the world. The absence of timely
detection of contamination compounds this issue, and the need for real-time contamination that
can be used to dynamically map microbe transport is an unnecessary obstacle in the path to
protecting our surface waters. This paper outlines the ability to conjugate microbe-capturing
primary antibodies to a versatile material such as a hydrogel, as well as the method to detect
primary antibody conjugation. The method of conjugating – using an intermediate cationic
molecule that is adsorbed by the hydrogel – sets the foundation for a new technological platform
for attaching useful molecules or tools to hydrogels.
1
Introduction
Microbe Contamination Detection
Water is perhaps the most precious resource on Earth, as people across the world struggle daily with
inadequate access to and protection of clean water. It is easy to associate the issue of water quality with
third-world countries, but the protection of water resources is an emerging political, economical,
environmental, and health-related issue in the United States. Waterborne pathogens are one of the
leading causes of water contamination in the U.S. Approximately 93,000 miles of rivers and streams in
our country have elevated bacterial levels (EPA 2000). These pathogens cause sickness and sometimes
death in vulnerable populations. The CDC estimates that over 900,000 illnesses and as many as 900
deaths are caused by waterborne pathogens each year (CDC 2008).
The problem of water contamination is compounded by our current inability to detect waterborne
pathogens in a timely manner. The predominant methods of detection of these pathogens are timeintensive, usually involving a 24-hour incubation period as part of the detection mechanism. This makes it
nearly impossible to have a true grasp on when and where surface waters are contaminated with harmful
pathogens. Therefore, there is a critical need to develop methods for microbial detection in real- or nearreal time, so that adequate measures can be implemented to reduce human exposure to contaminated
waters. The experiments outlined in this paper prove that hydrogels are capable of attaching to primary
antibodies for Escherichia coli (E. coli) and cryptosporidium. This capability may be used in an effort to
create a mobile and versatile biosensor.
Hydrogels
Hydrogels are hydrophilic polymer networks that consist primarily of water. The hydrogels
synthesized in these experiments were poly(N-isopropylacrylamide) (PNIPAM) hydrogels that use
Laponite-XLG as a polymer cross-linker. These particular hydrogels undergo a dramatic volume phase
transition above approximately 33 oC (Arora, et. al. 2009). The Laponite cross-linker has a negative
charge, which allows them to adsorb cationic molecules out of solution (Thomas, et. al. 2011; Gregory,
et. al. 2013). Past experiments have utilized this property as a mechanism for separations (Thomas, et.
al. 2011) or for remote-controlled volume phase transition induction (Gregory, et. al. 2013). Additionally,
non-PNIPAM hydrogels have been shown capable of binding to primary antibodies (Massad-Ivanir, et. al.
2010). Our experiments combine these two capabilities by allowing the hydrogels to bind specific cationic
molecules, which contain the functional groups necessary for primary antibody conjugation.
Experimental Methodologies
PNIPAM Hydrogel Synthesis
Glass tubes of approximately 1 cm in length with an inner diameter of 1.2 mm were made
hydrophobic by submerging them in solution of distilled water, 1.0% volume acetic acid (Sigma Aldrich),
and 0.5% volume dimethoxysilane (Sigma Aldrich). After the tubes were allowed to soak in the solution
for approximately 15 minutes, the tubes were removed and dried under vacuum in a vacuum oven at 250
oC until dry (approximately 30 minutes).
The protocol for synthesizing PNIPAM hydrogels is also described elsewhere (Arora, et. al. 2009). 40
mg of Laponite-XLG (Southern Clay) was added to 28.5 mL of distilled water and stirred for approximately
30 minutes until the solution returns to clear. 3.5 g of PNIPAM (Sigma Aldrich) is added to the solution
and is stirred for approximately 15 minutes until the solution returns to clear. A solution of 30 mg of
potassium persulfate dissolved in 1.5 mL of distilled water was added to the stirring PNIPAM and water
solution, followed immediately by 24 μL of N,N,N’,N’-tetramethylethylenediamine. The hydrophobic glass
tubes were added to the resulting solution, which was then allowed to polymerize for 24 hours at 50 oC.
2
After polymerization, the cylindrical hydrogels were removed from the glass tubes and allowed to hydrate
in distilled water for 24 hours at room temperature.
Acriflavinium Chloride Adsorption
PNIPAM hydrogels were submerged in a 5 mg mL-1
solution of acriflavine (Sigma Aldrich, 33% acriflavinium
chloride) and distilled water for 5 minutes (Figure 1).
Longer exposure to acriflavine dye would result in
degradation of the hydrogel. After dye adsorption, the
hydrogels were rinsed with and submerged in fresh
distilled water solutions repeatedly until the un-adsorbed
dye had been removed from the hydrogel.
Hydrogel
Figure 1 Representation of cylindrical
hydrogel submersion in an acriflavine
dye and water solution. Acriflavine dye
contains charged acriflavinium chloride
molecules (shown) which are adsorbed
by the hydrogel
Primary Antibody Attachment
The primary antibody attachment protocol was designed based on protein crosslinking techniques
from Hermanson (2008). Primary antibodies for E. coli (KPL) were suspended in PBS 1x to a 20 μg mL-1
concentration. One mL aliquots and 10 μL of glutaraldehyde were added to a separate vial. After 10
seconds, a hydrogel of approximately 1
Hydrogel
cm of length and 4 mm in diameter that
had adsorbed acriflavinium chloride was
added to the vial and allowed to react on a
NH2
NH2
nutator with the glutaraldehyde and
NH2
primary antibodies for 2 hours at 4 oC.
NH2
After the primary antibody conjugation
was complete, the hydrogels were place
in a 1:9 horse serum to PBS 1x solution
and allowed to rinse on a nutator for 30
minutes. Three subsequent equivalent
rinses were performed, after which the
hydrogel was placed in fresh PBS 1x and
Figure 2 Representation showing a primary antibody
kept refrigerated.
being conjugated to a hydrogel that has adsorbed
acriflavinium chloride by amine-to-amine chemistry.
This protocol was repeated as stated
The black line indicates the unknown mechanism
for
the attachment of Cryptosporidium
(Hermanson 2008) of amine chemistry performed by
(KPL) primary antibodies.
the homobiofunctional crosslinker glutaraldehyde
Primary Antibody Attachment Verification
The secondary antibody (KPL) to the primary E. coli antibodies, which were labeled with Alexa 647
fluorescent dye molecules, were added to PBS 1x in a 1:500 secondary antibody to PBS concentration.
The primary antibody-functionalized hydrogels were exposed to a 1 mL aliquot of the secondary antibody
solution on a nutator for 3 hours at room temperature, with minimal exposure to light. After the secondary
antibody exposure, the hydrogels were placed in clean PBS 1x and allowed to rinse on the nutator for 30
3
minutes. After rinsing, the hydrogels were placed in fresh PBS 1x. Three subsequent rinses were
performed, after which the hydrogel was placed in fresh PBS 1x and kept refrigerated and away from
light.
The protocol was repeated as stated for the verification of
Cryptosporidium primary antibodies. Secondary antibodies
(Abcam) that correspond to the Cryptosporidium primary
antibodies were used in place of the secondary antibodies for E.
coli antibody verification.
Four samples were imaged using a 3024 Nikon A1R MultiPhoton Upright Confocal microscope. Three of the samples were
cross-sections of hydrogels that went through the acriflavinium
chloride adsorption, E. coli or Cryptosporidium primary antibody
attachment protocols and were exposed to the corresponding
fluorescent-labeled secondary antibodies. The other three samples
were the respective controls for the three antibody-conjugated
hydrogels. The controls were a necessity to ensure that the
presence of secondary antibodies was indicative of primary
antibody attachment and not due to secondary antibodies sticking
to the structure of the hydrogel or acriflavinium hydrochloride. A
summary of the imaged samples can be found in Table 1.
Hydrogel
Alexa 647 label
Figure 3 Representation of the
mechanism for primary antibody
attachment, which is the specific
binding of fluorescent-labeled
secondary antibodies
Table 1: Summary of four samples imaged for primary antibody verification
Sample 1
Sample 2
Sample 3
Sample 4
Acriflavinium
Yes
Yes
Yes
Yes
Chloride
Goat Anti-E.
Primary Antibody
None
None
Mouse Anticoli
Cryptosporidium
Secondary
Donkey
Donkey
Goat AntiGoat AntiAntibody
Anti-Goat
Anti-Goat
Mouse
Mouse
Results
Acriflavine Adsorption
Hydrogels have previously been shown to adsorb
water-soluble cationic dyes out of solution. (Thomas et.
al. 2011; Gregory et. al. 2013). Acriflavine dye contains
acflavinium chloride (Figure 1), which has a cationic
quaternary amine in its chemical structure. The
hydrogels effectively adsorbed the acriflavine dye out of
solutions. The acriflavine dye solution contains
approximately 33% of the cationic molecule, resulting in
leaching of molecules that are not adsorbed by the
hydrogel. However,
after washing the hydrogels
repeatedly to remove un-adsorbed dye, a sufficient
amount of acriflavinium chloride is adsorbed in to the
structure of the hydrogel (Figure 4).
Figure 4 PNIPAM hydrogels; one has
not adsorbed acriflavine dye (top), one
after acriflavine dye adsorption (bottom)
4
Primary Antibody Attachment
Table 2: Results from Primary Antibody Attachment and Verification Experiment
Sample 1
E. Coli Antibody
Control
Sample 2
E. Coli Antibody
Sample
The picture on the left shows
fluorescence as excited by a
488 nm laser. The green
fluorescence is indicative of
acriflavinium
chloride
adsorption. The picture on
the right shows the lack of
fluorescence excited by a
640 nm laser (brightness
enhanced by 50%). The lack
of fluorescence at 640 nm
shows that the Alexa-647labeled secondary antibodies
are not binding to the sample
due to the structure of the
hydrogel or the presence of
acriflavinium chloride.
The picture on the left shows
fluorescence as excited by a
488 nm laser. The green
fluorescence is indicative of
acriflavinium
chloride
adsorption. The picture on
the right shows the presence
of
Alexa-647-labled
secondary antibodies as
excited by a 640 nm laser
(brightness enhanced by
50%). The presence of
secondary
antibodies
in
Sample 2 but not in Sample 1
shows that the secondary
antibodies are present due to
the successful conjugation of
primary antibodies.
5
Sample 3
Cryptosporidium
Control
Sample 4
Cryptosporidium
Sample
The picture on the left shows
fluorescence as excited by a
488 nm laser, which is
indicative of acriflavinium
chloride adsorption. The
picture on the right shows the
lack of fluorescence excited
by a 640 nm laser
(brightness enhanced by
50%). The lack of
fluorescence at 640 nm
shows that the Alexa-647labeled secondary antibodies
are not binding to the sample
due to the structure of the
hydrogel or the presence of
acriflavinium chloride.
The picture on the left shows
fluorescence as excited by a
488 nm laser. The green
fluorescence is indicative of
acriflavinium
chloride
adsorption. The picture on
the right shows the presence
of
Alexa-647-labled
secondary antibodies as
excited by a 640 nm laser
(brightness enhanced by
50%). The presence of
secondary
antibodies
in
Sample 2 but not in Sample 1
shows that the secondary
antibodies are present due to
the successful conjugation of
primary antibodies.
Discussion
The results of the experiment were successful and have two-fold ramifications. First, the ability to
functionalize the PNIPAM hydrogels with the ability to capture microbials is a step towards creating a
mobile hydrogel-based biosensor. Hydrogels have previously been shown to capture E. Coli (MassadIvanir, et. al. 2010), but this is the first time that PNIPAM, Laponite cross-linked hydrogels have been
functionalized to do so. This is important because these hydrogels have durability (Yeghiazarian 2005)
and are capable of mobility in both constrained and unconstrained environments via peristaltic locomotion
(Gregory 2013), which increases this particular hydrogel’s propensity for usage as a mobile biosensor
capable of gathering detection data useful for dynamic mapping of microbe transport. Furthermore, our
method of primary antibody conjugation was direct – conjugating antibodies with the homobiofunctional
crosslinker glutaraldehyde to dye molecules adsorbed into the hydrogel. This is an alternate method to
those performed by Massad-Ivanir et. al. (2010).
Additionally, this experiment has ramifications across the increasingly popular field of hydrogels. In
functionalizing the hydrogel by conjugating antibodies to an adsorbed molecule, we have created a new
6
technological platform in which any number of molecules or devices may be attached to a hydrogel by
first adsorbing a cationic dye with functional groups conducive to attaching the respective molecule or
device. Outside the field of biosensors, there are opportunities to increase the usage of hydrogels as a
soft biomedical soft tool used for targeted drug delivery or cavity exploration.
Acknowledgements
We would like to thank the National Science Foundation’s Research Experience for Undergraduates
program (NSF Type-1 STEP Grant, Grant ID No.: DUE-0756921) as well as the National Science
Foundation’s “EAGER: Monitoring Our Nation’s Waters – Towards a Swimming Biosensor to Dynamically
Map Microbial Contamination” grant (NSF CBET-1248385) for financial support of this project.
Additionally, we would like to thank the Cincinnati Children’s Hospital and Medical Center Imaging
Center, the director of the imaging center Dr. Matthew Kofron, and the manager of the imaging center Mr.
Michael Muntifering for the access to and their assistance with fluorescent imaging on the 3024 Nikon
A1R Multi-Photon Upright Confocal microscope.
References
1.
U.S. Environmental Protection Agency (EPA). 2000. National water quality inventory: 1998
Report to Congress. EPA-841-R-00-001. 413.
2.
<http://www.cdc.gov/nceh/vsp/training/videos/transcripts/water.pdf>
3.
<http://www.antimicrobialresistance.dk/data/images/protocols/e%20coli%20methods.pdf>
4.
Arora, H., Malik, R., Yeghiazarian, L., Cohen, C., and Wiesner, U. (2009). “Earthworm Inspired
Locomotive Motion from Fast Swelling Hybrid Hydrogels.” Journal of Polymer Science: Part A:
Polymer Chemistry, 47, 5027–5033.
5.
Thomas, P. C., Cipriano, B. H., and Raghavan, S. R. (2011). “Nanoparticle-crosslinked hydrogels
as a class of efficient materials for separation and ion exchange.” Soft Matter, 7(18), 8192–8197.
6.
Gregory, J., Riasi, M. S., Cannell, J., Arora, H., Yeghiazarian, L., Nistor, V. (2013). “RemoteControlled Peristaltic Locomotion in Free-Floating PNIPAM Hydrogels.” Advanced Materials,
Submitted for Publication.
7.
Massad-Ivanir, N., Shtenberg, G., Zeidman, T., and Segal, E. (2010). “Construction and
Characterization of Porous SiO2/Hydrogel Hybrids as Optical Biosensors for Rapid Detection of
Bacteria.” Advanced Functional Materials, 20(14), 2269–2277.
8.
Hermanson, G. T. (2008). “Homobiofunctional Crosslinkers.” Bioconjugate Techniques, 2nd
Edition. Thermofisher Scientific, Rockford, IL, 234-275.
9.
Yeghiazarian, L., Mahajan, S., Montemagno, C., Cohen, C., and Wiesner, U. (2005). “Directed
Motion and Cargo Transport Through Propagation of Polymer-Gel Volume Phase Transitions.”
Advanced Materials, 17(15), 1869–1873.
7