ELECTROSPUN NANO-FIBERS AS BIOSEPARATORS: CHARGE BASED
SEPARATION OF ESCHERICHIA COLI FROM LIQUID SAMPLES
A thesis
Presented to the Faculty of the Graduate School
of Cornell University
in Partial Fulfillment of the Requirements for the degree of
Master of Engineering
by
Marissa Agustin
January 2011
© Marissa Agustin 2011
ABSTRACT
Biosensors for pathogen detection have the potential to prevent and identify
health hazards in environmental samples and food samples. However, in order to detect
pathogens in complex samples, concentration and separation of the bacteria from the
sample matrix is necessary. Obtaining rapid accurate results is essential for detecting
foodborne pathogens, as traditional methods can take several days to produce a result.
Sample preparation is time consuming and usually performed off-chip. New technologies
such as lab-on-a-chip platforms aim to integrate several laboratory operations onto a
single chip. Electrospun nanofibers can be incorporated into microfluidic channels and
can be functionalized with various bio-recognition elements. The electrospinning process
uses an electrical charge to draw out thin continuous polymer threads with diameters
ranging from 10-1000nm. These nanofibers are therefore ideal for use in filtration
because they provide a higher surface area as compared to traditional membranes.
Functionalized nanofibers carrying positive and negative charges were spun by
the Frey Group at Cornell University. The fibers were investigated for their ability to
separate E.coli from buffer samples and actual apple juice samples. This separation is
based on the knowledge that E.coli and gram negative bacteria have a strong net negative
charge at neutral pH and a net positive charge at low pH caused by deprotonation and
protonation of carboxyl and ammonium groups on the cell surface1. Samples buffers at
pH7 and pH4 were used in this study because the pH of apple juice is typically around 3
or 4. Filtration was evaluated using two methods 1) by collecting cells at the channel
outlet, plating on Trypto Soy Agar (TSA) and counting colonies the next day and 2) by
staining the cells with Syto9 green fluorescent dye and capturing digital images using RS
CoolSNAP camera and RS Image software. The images were then analyzed in ImageJ to
measure fluorescence intensity in the channel, high fluorescence correlating to the
presence of E.coli on the fibers.
In the first method, positive fibers were shown to retain 98 ± 1.6% of E.coli that
passed through the microfluidic channels whereas negative fibers only retained 35 ±
1.1%. Unexpectedly, in the empty channel only 53 ± 4.4% of cells were recovered at the
outlet. Since there are no fibers in the channel, close to 100% recovery would be
expected however, cell losses could be attributed to cells adhering to the hydrophobic
channel and tubing surfaces or cells becoming trapped at the inlet or outlet holes. Coating
the channel and tubing with 1% BSA prior to experiments increased the recovery at the
outlet to 83 ±15 % of the cells.
In the second method, epifluorescence measurements provided a less quantitative
result but allowed for visualization of E.coli on the fibers. E.coli in pH7 buffer was
filtered onto positive fibers producing a strong fluorescence. The cells were eluted by
shifting pH conditions by introducing a pH4 washing buffer producing an 84% drop in
fluorescence intensity. E.coli in pH4 buffer attached better to negative fibers producing
average fluorescence intensity 1.6 times higher than the channels with positive fibers.
The results were repeated with apple juice spiked with E.coli producing similar results.
The negative channels produced fluorescence intensity 2 times higher than in the positive
channels.
These results show the potential for electrospun nanofibers to be used on-chip as
bioseparators. The filtration investigated here is non-specific with the purpose of isolating
bacteria from a sample matrix which might be an environmental sample or a food sample.
Once the bacteria are immobilized on the fibers, they can either be detected using
fluorescently tagged antibodies or eluted to be detected further downstream. Due to time
constraints and availability of fibers, some experiments in this study were not repeated in
triplicate. Further studies should be done to confirm the results. In order to design
nanofiber mats for different filtration applications, further experiments need to be done to
correlate spinning time to fiber pore size and fiber mat density.
ACKNOWLEDGEMENTS
I am grateful for all the guidance that my advisor, Professor Antje Baeumner, has
given me in this project. This thesis would not have been possible without her knowledge
and expertise. I would also like to thank her for the kindness and patience she has shown
me as my undergraduate advisor, research advisor and teacher throughout the four and
half years that I have been at Cornell.
I would like to thank Lauren Matlock-Colangelo for the long hours she spent in
the CNF making electrodes, for teaching me how to use the lab equipment and how to
fabricate PMMA devices. I would like to thank Christian Willrodt and Lauren for their
help and being there to bounce ideas off. I would like to thank Dr. Daehwan Cho and
Lauren for spinning the nanofibers I needed for my experiments.
I would like to thank the Department of Biological and Environmental
Engineering staff, faculty and my professors who have been a tremendous help and from
whom I have learned so much.
I would like to give a big hug to all my friends who stayed at Cornell for MEng or
PhD degrees and toughed the semester out with me. Thanks to Collegetown Bagels for all
the coffee, warmth, and pastries and for allowing me to sit in your store for hours on end
analyzing data and writing this thesis. Finally, I would like to thank my parents, my
brother, Michael and my sisters, Angela, Rebecca and Rosenda, for all the love and
support they have shown me.
BIOGRAPHY
Marissa Agustin is from Scarsdale, New York. She has received both a Master of
Engineering and a Bachelor of Science in Biological Engineering from Cornell
University. Her interests are in microfluidics, biosensors, biological process engineering,
and food safety. She is certified as an Intern Engineer by the State of New York having
passed the Fundamentals of Engineering Exam in April 2010. She is a member of the
Cornell Iota Beta Chapter of Alpha Epsilon, the national honors society of Agricultural,
Food and Biological Engineering. Her hobbies include playing flute, Filipino folk
dancing and making her own fermented food products.
LIST OF FIGURES
Figure 1: Top view of device channel design
Figure 2: Microfluidic Device Fabrication Scheme
Figure 3: Electrospinning Setup
Figure 4: Size distribution of particles in apple juice
Figure 5: Volume Fraction of particles in apple juice according to diameter
Figure 6: Cell counting: preliminary results of nanofiber filtration.
Figure 7: Cell counting: nanofiber filtration of E.coli
Figure 8: Fluorescent measurements for filtration of pH7 samples
Figure 9: Fluorescent images for positive fibers washed with pH4 buffer
Figure 10: Fluorescent images for positive fibers washed with pH7 buffer
Figure 11: Comparison of filtration of pH4 buffer samples and apple juice samples
LIST OF ABREVIATIONS
ATCC
American Type Culture Collection
BSA
Bovine Serum Albumin
CDC
Center for Disease Control
CFU
Colony forming units
CNF
Cornell NanoScale Science and Technology Facility
DI
Deionized
DLS
Dynamic Light Scattering
DMSO
Dimethyl sulfoxide
DNA
Deoxyribonucleic acid
IMS
Immuno-magnetic separation
NASBA
Nucleic acid sequence based amplification
PBS
Phosphate Buffered Saline
PMMA
Poly-methyl methacrylate
Polybrene
Hexadimethrine bromide
PolyMA
Polymethyl vinyl ether-alt-maleic anhydride
PVA
Polyvinyl Alcohol
RT-PCR
Real time polymerase chain reaction
TSA
Trypticase soy Agar
TSB
Trypticase soy Broth
UF
Ultrafiltration
UV
Ultraviolet
UV/O3`
Ultraviolet ozone treatment
TABLE OF CONTENTS
ABSTRACT.............................................................................................................................. 3
LIST OF FIGURES .................................................................................................................. 7
LIST OF ABREVIATIONS ..................................................................................................... 8
1.0 INTRODUCTION ............................................................................................................ 11
1.1 Motivation ..................................................................................................................... 11
1.2 Separation Methods ...................................................................................................... 12
1.3 Electrospun Nanofibers ................................................................................................. 13
1.4 Microfluidic Devices .................................................................................................... 13
1.5 Sample Matrix............................................................................................................... 14
2.0 DESIGN ............................................................................................................................ 16
3.0 MATERIALS AND METHODS ...................................................................................... 18
3.1 Materials ....................................................................................................................... 18
3.2 Bacterial Culture ........................................................................................................... 18
3.3 Dynamic Light Scattering ............................................................................................. 19
3.4 Device Fabrication ........................................................................................................ 19
3.5 Electro-spinning ............................................................................................................ 21
3.6 Method 1: Quantitative Cell Counts ............................................................................. 22
3.7 Method 2: Epifluorescent Microscopy .......................................................................... 23
4.0 RESULTS ......................................................................................................................... 24
4.1 Apple Juice Dynamic Light Scattering ......................................................................... 24
4.2 Results: Quantitative Cell Counts ................................................................................. 25
4.3 Results: Epifluorescent Microscopy ............................................................................. 28
5.0 CONCLUSION AND FUTURE WORK ......................................................................... 32
1.0 INTRODUCTION
1.1 Motivation
The most recent statistics on food borne illness published in 2011 by the CDC
estimate that there are 47.8 million cases, 127,839 hospitalizations, and 3,037 deaths per
year in the United States caused by foodborne illnesses2,3. These numbers are lower than
the statistics reported in 19994; however, because of differences in methodology the two
studies cannot be compared. Therefore it also cannot be concluded that food quality and
safety in the US has improved. In fact, the new data indicates that cases of foodborne
illness are still quite high. To ensure that health and safety standards are being met, there
is a need for rapid pathogen detection methods that are both sensitive and specific.
However, research efforts aimed at detection of food-borne pathogens often focus
solely on identification of the bacteria and overlook the preliminary sample preparation.
The processing steps used for sample concentration and separation are important because
food matrices are both diverse and complex. Food samples usually contain very low
levels of pathogenic organisms and may contain a wide range of background microflora.
In addition, particulates in food may shield microorganisms or inhibit reactions necessary
for DNA based detection. In the food industry, the gold standard for pathogen detection is
enrichment culture. This method relies on availability of media that will selectively
enhance growth of a target organism while inhibiting growth of other bacteria that may
be present in the sample. Typically, amplification of cell numbers to levels detectable by
standard plate count can take over 24 hours. Consequently, producers experience
economic losses because food products need to be held until lab tests are completed.
Furthermore, this method does not give direct quantitative information about the original
level of bacteria in the food sample. To address these problems, rapid tests and sensors
that use real time polymerase chain reaction (RT-PCR), nucleic acid sequence based
amplification (NASBA), immunoassays or amperometric methods have been developed
for detection of pathogens 5,6. Molecular based detection methods are fast, accurate and
sensitive; however, they can only process sample volumes of a few µL. This is a problem
because standard food sampling usually requires greater than 25g or 10mL per sample
because of the low levels of pathogenic bacteria in foods. Compounds present in the food
sample or enrichment media may also interfere with enzymes and antibodies used in
molecular based detection methods. In order for these more advanced technologies to be
adopted for quality control in the food industry, methods must be developed that are
capable of both reducing sample volumes and separating the target analyte from the food
matrix.
1.2 Separation Methods
Existing separation methods for isolation of bacteria from food samples can be
grouped into two categories: physical methods and adsorption methods. Summaries of
different types of physical and adsorption methods used in the food industry have been
published in microbiology and food science journals. The more commonly used methods
include centrifugation, filtration, and immune-magnetic separation (IMS) 7,8,9.
Centrifugation and filtration are able to effectively concentrate bacteria but also end up
concentrating food particles along with the target analyte. The removal of food debris
requires multiple wash and centrifugation steps and, in the case of filtration, the debris
will eventually foul the filtration membrane and decrease flux rates. IMS is an attractive
method because it is capable of concentrating samples, removing impurities and isolating
specific bacterial strains from food matrices. However, IMS is expensive because of the
cost of monoclonal antibodies and because the method can only process relatively small
sample volumes. Food debris may become trapped between the magnetic beads and
particles in the food may interfere with binding events. Therefore, IMS may require
multiple wash and magnetic collection steps which is time consuming and hard to
integrate into an on-line system.
1.3 Electrospun Nanofibers
Recently, there has been increased interest in electrospun nanofibers which
exhibit properties attractive for filtration, as well as a wide range of other applications in
the food, biotechnology and biomedical industries10. Nanofibers with small diameters,
10-1000nm, can be spun uniformly into non woven mats and across microfluidic
channels. These fibers have high surface area to volume ratios, porosity and mechanical
strength7. Research has also shown that it is possible to functionalize these nanofibers by
incorporating compounds into the electrospinning solution11. Functionalization allows for
selectivity based on chemical properties of the target analyte in addition to selectivity
based on size. The pores formed by these nanofiber mats can range from 0.16 to 8.03µm
depending on fiber diameters12. In comparison E.coli cells are about 0.5µm in diameter
and 1-2µm long. Integration of membranes into microfluidic devices has substantial
applications for sample preparation, separation, purification, biosensing, etc.13.
1.4 Microfluidic Devices
Fabrication methods, well established in the semiconductor industry utilize glass
and silicon as substrates. For biological applications, glass has useful properties but tends
to be expensive. Polymer chips are increasingly being used for microfluidic devices
because they are transparent, disposable and relatively low cost14. The fabrication
processes for microfluidic devices made using polymer substrates have been described
previously15, 16. Microfluidic channels were fabricated in poly-methyl methacrylate
(PMMA) using hot embossing with a copper master. PMMA requires surface
modification with UV-ozone (UV/O3) treatment to allow for successful low temperature
thermal bonding. UV/O3 exposure inserts oxygen containing functional groups into the
polymer surface decreasing hydrophobicity17.
1.5 Sample Matrix
Commercial juices are a feasible starting point for separation and concentration of
actual food samples because they contain few solid particles and are not very viscous.
Recently, attention has been focused on monitoring the processing of fruit juices.
Contamination with Escherichia coli O157:H7 is usually associated with ground beef and
dairy products, however, between 2002 and 2005, six juice-associated outbreaks of
illness were reported by the CDC and five out of the six were linked to apple juice18. The
source of E.coli contamination is usually feces and manure which can be introduced to
fruits when they fall on the ground. Fruits pressed with the skins on can then lead to
contamination of the juice. To my knowledge, nanofibers spun into microfluidic channels
have not yet been applied to sample concentration for microbial testing of beverages. One
study investigated electrospun nanofiber membranes for clarification of apple juice19. In
that study, a dead-end filtration was performed using a large, 63mm diameter, poly
(ethylene terephthalate) nanofiber mat. The nanofiber filtration produced similar quality
apple juice to product obtained using a traditional cellulose UF membrane. In addition,
the composition of dissolved solutes (phenolic compounds, sugars and organic acids) in
the nanofiber filtered apple juice was almost the same as the composition of the unclarified control juice. Phenolic compounds are known to interfere with PCR detection of
E. coli 0157:H7 in juice samples20. Therefore the fact that these compounds passed
through the nanofiber mat is promising for sample concentration. Ideally, the nanofiber
mat should retain the target bacteria while allowing impurities that would interfere with
pathogen detection to pass through the fibers.
Apple juice composition will vary with variety of the fruit, climate, processing
conditions and storage. Typically the pH is between 3.0-3.8. Sugars make up about 714% of soluble components in apple juice, malic acid is present at a concentration of
0.5% and soluble proteins, phenolic compounds and pectin make up 120-500ppm
weight/volume of apple juice. Other constituents include minerals, potassium, calcium,
and esters21. The natural microflora of apple juice is comprised of mostly yeasts and
molds. However, contamination with E.coli and Salmonella has been associated with
unpasteurized apple juice.
2.0 DESIGN
Previous work done in the Baeumner lab has shown that electrospun nanofibers
can be successfully incorporated into the microfluidic channels of PMMA devices using
thermal bonding. Polyvinyl alcohol (PVA) nanofibers, provided by the Frey group in the
department of Fiber Science and Apparel Design at Cornell University, were tested for
their effectiveness for charged based separation. Negatively charged fluorescent
liposomes have been found to adhere to positively charged fibers. The liposomes can be
observed by placing the PMMA devices under an epifluorescence microscope and
measuring fluorescence intensity over time.
In this study functionalized nanofibers were tested for a different application.
E.coli and gram negative bacteria are also known to have a negative charge at neutral
pH1 but bacterial cells are much larger than liposomes. The objectives of this study were
to 1) provide a proof of concept that E.coli can be captured by functionalized nanofibers
2) visualize E.coli on the fibers using fluorescent staining 3) determine the conditions to
elute attached bacteria from the fibers and 4) evaluate the effectiveness of nanofiber
filtration of actual apple juice samples spiked with E.coli.
The filtration was evaluated using two methods 1) by collecting cells at the
channel outlet, plating on TSA and counting colonies the next day and 2) by staining the
cells with Styo9 and using epifluorescent microscopy and an imaging software to
measure fluorescence intensity in the channel. Fluorescence measurements offer a
qualitative measurement of binding however, E.coli can be quantified directly using
standard plate counts. In method 1, the number of cells collected at the outlet can give a
quantitative measure of how many cells pass through the fibers and because the initial
concentration of cells is known, how many cells were retained by the fibers. E.coli
samples in pH7 buffer were filtered through channels with no fibers, positive fibers and
negative fibers.
In method 2, the filtration effectiveness of channels was determined by the
fluorescence intensity in the channel. Channels with positive fibers and negative fibers
were compared for E.coli spiked samples in pH7 buffer and pH4 buffer. The experiments
done in pH4 buffer were also compared to experiments with actual apple juice samples
(ph 3.9) spiked with E.coli. After pumping the sample through the channel for 20
minutes, the syringe was switched and the fibers were washed with a pH4 wash buffer, a
pH7 wash buffer or apple juice depending on the experiment.
Originally the microfluidic device design consisted of a single channel 30mm in
length, 1mm wide and 50μm deep. The second chip design consisted of 4 parallel
channels with the same dimensions as in the original chip. Tubing was cut to a length
such that the wash buffer would hit the channel exactly 20 minutes after the syringe was
switched if the flow rate was kept at 1μL/min.
3.0 MATERIALS AND METHODS
3.1 Materials
Syto 9 ® Green Fluorescent Nucleic Acid Stain 5mM solution in dimethyl sulfoxide
(DMSO) was purchased from Invitrogen (Eugene , OR). Trypto soy agar (TSA) and
Trypto Soy Broth (TSB) were purchased from BD Biosciences (Sparks, MD). Apple
juice (Tops Brand) was purchase from the local supermarket (Ingredients: apple juice
concentrate, water, ascorbic acid). Stainless steel blunt needles with Luer polypropylene
hub 22 Gauge were purchased from VWR. Tygon Micro Bore PVC tubing with inner
diameter 0.02 in and outer diameter 0.06 in were purchased from VWR.
3.2 Bacterial Culture
Non pathogenic E.coli K-12 strain ATCC 25922 was obtained from Prof. Randy Worobo
at the Dept. of Food Science and Technology, Geneva, NY. The culture was streaked for
isolation on plates containing 40g/L Trypto Soy Agar (TSA) pH 7.3 and incubated at
37°C for 20 hours. A 40g sample of TSA contains approximately 15g pancreatic digest of
casein, 5 g enzymatic digest of soybean meal, 5g sodium chloride, and 15g agar. The
media was prepared in a 1L Erlenmeyer flask, 20g TSA powder was dissolved in 0.5L DI
water and autoclaved at 121°C for 35 minutes. The media was cooled to 60°C and poured
into petri plates. Plates were left on the bench overnight to cool and then stored at 5°C for
later use. Liquid cultures were grown in liquid media containing 30g/L Trypto Soy Broth
(TSB), final pH 7.3. A 30g sample of TSB contains approximately 17g of pancreatic
digest of casein, 3g enzymatic digest of soybean meal, 2.5g dextrose, 5g sodium chloride,
and 2.5 g of dipotassium phosphate. The liquid media was prepared by dissolving 6g of
TSB in 200mL of DI water. 10mL aliquots of TSB were dispensed into test tubes and
then autoclaved at 121°C for 35 minutes.
To prepare samples for experiments, a single colony of E.coli was transferred into a test
tube with 10mL TSB (30g/L) and incubated for 20 hours in a Lab-Line Orbit Environ
Shaker (Melrose, IL) at 150rpm. To determine the initial concentration of cells in the
broth, 100µL samples of the culture dilutions 104 - 106 in PBS were pipetted and spread
onto TSA plates and incubated overnight at 37°C. Colonies were counted the following
day and the initial concentration in CFU/mL was calculated from this number. CFU =
#colonies x 10n where n = the dilution factor. It was found that consistently, after 20
hours, the culture contained a concentration of approximately 109 CFU/mL. The culture
was then serially diluted in phosphate buffered saline (PBS) to desired concentrations for
experiments.
3.3 Dynamic Light Scattering
DLS analysis was performed using a 90 Plus Nanoparticle Size analyzer with a Peltier
temperature control system (Brookhaven Instruments, Holtsville, NY). 40mL samples of
20%, 50% and 100% juice solutions in UF water were prepared. Measurements were
taken at a 90° angle. A cuvette was filled with the sample and inserted into the machine
for a reading. The temperature was set to 20°C., dust cut-off was set at 95% to increase
the signal retention because of preliminary measurements that showed particles in the μm
range. Apple juice viscosity was assumed to be that of water, 1.002 cP and particles were
assumed to be perfect spheres. Refractive index for each of the samples was measured
using a digital refractometer (Misco Products Division, Cleveland, OH) and inputted into
the BIC software.
3.4 Device Fabrication
Microfluidic devices were fabricated in PMMA using hot embossing and UV assisted
thermal bonding. Pieces of PMMA approximately 50mm x 50 mm were cut using a band
saw (Ryobi Tools).
Figure 1: Top view of channel design
The copper master template was fabricated by Lauren Matlock-Colangelo in the CNF
using photolithography, wet etching and electroplating to produce the desired raised
pattern12. Microfluidic channels were embossed into the PMMA by sandwiching the
PMMA chip between the copper master and another copper plate. The PMMA copper
sandwich was warmed on the hot press (Fred S. Carver Inc., Summit NJ) at 120°C for 5
minutes and then 5000lb of pressure was applied for 10 minutes at 120°C. Inlet and
outlet holes with a diameter of 0.8mm were drilled using the drill press (Ryobi Tools).
PMMA chips with embossed channels were sonicated (Aquasonic 75D VWR Scientific)
at room temperature for 10 min in 2-propanol.
The PMMA chips with the embossed channels and the PMMA chips with the
nanofiber mats were then exposed to UV/O3 treatment to activate the polymer surface
and lower the glass transition temperature of the PMMA. The UVO Cleaner (Jelight
Company Inc, Mason Irvine, California) was turned on and warmed up for 10min on and
the oxygen flow rate set to 1.0L/min. The oxygen flow rate was lowered to 0.5L/min for
the treatments. PMMA chips were placed channel or fiber side up on the tray in the UVO
Cleaner and PMMA chips with channels were treated for 10 minutes and PMMA chips
with nanofibers were treated for 6 minutes. Exhaust time was set to 2 minutes.
The two PMMA chips were then sandwiched between two copper plates and
bonded for 10 minutes at 80°C under 4000lb of pressure. Tubing was cut ,super-glued
into the inlet and outlet holes and allowed to dry overnight.
Figure 2: Fabrication Scheme (a) PMMA chip sandwiched between the copper master
template and another copper plate (b) Sandwich warmed on hot plate for 5 minutes at
120°C then placed under 5000lb pressure at 120°C for 10 minutes (c) PMMA chip with
embossed channels (d) nanofiber mat spun onto another PMMA chip (e) PMMA chips
with nanofiber mat and embossed channels placed between two copper plates and bonded
at 80°C for 10 minutes under 4000lb of pressure (e) final device with nanofibers enclosed
in microfluidic channels
3.5 Electro-spinning
Electrospun nanofibers were produced by Dr. Daehwan Cho from the Frey Group,
Cornell University Fiber Science Department and by Lauren Matlock-Colangelo from the
Baeumner Group, Cornell University Biological and Environmental Engineering
Department. Spinning dope solutions were prepared by dissolving 10 wt% PVA polymer
in DI water. Functional polymers, Polybrene and PolyMA, were added to the spinning
dope for positive and negatively charged nanofibers respectively. A 5 mL plastic syringe
with an 18 gauge needle connected to a pump was used to eject the spinning dope. A
voltage of 12-15kV was applied to the needle tip and fibers were collected on grounded
PMMA chips as shown in Figure 3.
Figure 3: Electrospinning Setup and PMMA collecting chip with gold electrode array.
The distance between the collector and the needle tip was 10-15 cm. Electrospinning was
performed at room temperature. Gold electrode arrays on the PMMA collecting chip
were necessary to direct the fibers to land on the chip and for the fibers to attach to the
PMMA surface. The electrode arrays were produced by Lauren Matlock-Colangelo in the
CNF. The process for patterning Au electrodes on PMMA using gold-thiol chemistry has
been described previously11.
3.6 Method 1: Quantitative Cell Counts
E.coli was grown overnight to a concentration of 109 CFU/ml and then diluted to 103
CFU/mL in PBS at pH7. A 100 µL sample containing approximately 100 CFU was
pumped into the device at 1μL/min. Effluent was collected at the outlet and plated on
TSA plates to determine the number of cells that passed through the device and
indirectly, how many cells were retained by the fibers. For blocking experiments, 1%
BSA was pumped through the channels at 1μL/min for 3 hours and then the solution was
flushed out by pumping air through the device.
3.7 Method 2: Epifluorescent Microscopy
E.coli was grown overnight to a concentration of 109 CFU/ml and then stained with
Syto9 green fluorescent nucleic acid dye with excitation 485 nm and emission 498 nm.
The E.coli cells were centrifuged and washed to remove the nutrient broth. A 1mL
aliquot of culture was centrifuged at 10,000rpm for 1 minute, the TSB was discarded and
the cells re-suspended in 1mL of the sample solution. The cells were centrifuged again,
the supernatant discarded and the cells re-suspended in 1mL of the sample solution.
Sample solutions tested were pH7 PBS buffer, pH4 acetate buffer and apple juice. Cells
were stained with Syto9 nucleic acid stain by adding 1µL Syto 9 for every 300µL of
undiluted cell culture. A 1:10 dilution in of the stained cells was used to provide a
concentration high enough to saturate the fibers but prevent clogging the channel inlet.
For the fluorescent measurements, a FITC 31001 filter (Chroma Technology) with
excitation 480 nm, emission 535 nm was used. Image acquisition took 3 seconds. The
microfluidic devices were placed underneath the microscope (Leica Microsystems,
Weltzar, Germany) for the duration of the experiment. Images were captured using RS
Image software every 5 minutes. The sample spiked with stained E.coli cells was pumped
through the device at 1μL/min for 20 minutes. The syringe was then switched to the wash
buffer, either a pH4 buffer, pH7 buffer, or apple juice. The wash buffer was pumped into
the device at 1μL/min. The tubing was cut such that the wash buffer would reach the
channel at around the 40 minutes from the start of the experiment. Images were analyzed
in ImageJ software. The mean gray value was used to determine fluorescence intensity.
4.0 RESULTS
4.1 Apple Juice Dynamic Light Scattering
Commercial apple juice is filtered to remove sediment however protein-polyphenol hazes
can develop in clarified fruit juices especially in juice where pectin content is high.22
Particles larger than 10μm suspended in apple juice may clog the tubing, channels or
fibers in microfluidic devices. Therefore, a DLS analysis was performed on pasteurized
clarified apple juice to determine whether filtration or centrifugation of the apple juice
would be needed DLS measures fluctuations in the intensity of scattered light caused by
random Brownian motion of particles in a solution. Figure 4 shows that there are 2
groups of particles present in apple juice. There are small particles less than 1μm in
diameter and large particles that range between 10-50μm in diameter.
120
Intensity
100
80
60
40
20
0
1
10
100
Diameter, nm
Figure 4: Size distribution of particles in apple juice.
1000
10000
The volume fraction of particles vs. particle diameter in the apple juice sample was
plotted in Figure 5. Number corresponds to the volume fraction which is the solids
volume divided by the total volume. The graph shows that the volume fraction of
particles with diameters ≤ 1nm was 80%. while the volume fraction of particles with
diameters between 10-50μm was very small, approximately 1%, the results confirm that
before testing actual apple juice samples, a pre-filtration or centrifugation would be
needed in case there are large particles present in the sample.
Figure 5: Graph of number (volume of solids/ total volume) of particles according to
particle diameter in apple juice samples. The bars represent the volume fraction of
particles at that diameter. For example, the selected bin (green bar) corresponds to the
volume fraction of particles with 1nm diameters. Particles with diameters between 1050μm make up approximately 1% volume fraction of the sample. The red line is a graph
of cumulative volume fraction vs. particle diameter. The cumulative number for particles
with diameters ≤ 1nm is represented on the graph as the green horizontal line and
indicates that approximately 80% of particles in the solution are ≤ 1nm in diameter.
4.2 Results: Quantitative Cell Counts
E.coli has a net negative charge at neutral pH1 therefore, in theory at these conditions
bacterial cells should adhere to positively charged fibers and pass through neutral fibers.
PBS buffer at pH7 was spiked with E.coli and filtered through channels with no fibers,
positively charged fibers and neutral fibers. A flow rate of 1μL/min was chosen to
prevent the fibers from being washed out of the channel and because no noticeable
deterioration of the fibers was observed at this flow rate. The preliminary results in
Figure 6 show that almost all of the cells were captured by the positive fibers. Only 55
cells were recovered at the outlet of the channel with positive fibers whereas there were
close to 1000 cells recovered at the outlet of the empty channel and at the outlet of the
channel with the neutral fibers.
1200
1000
CFU
800
600
400
200
0
Initial Cell
Concentration
No Fibers
Neutral
Positive
Figure 6: Preliminary results of nanofiber filtration. Cells were collected at the outlet and
plated on TSA. The sample volume was 1000μL and contained approximately 103 CFU
of E.coli. A flow rate of 1μL/min was used.
When negative fibers became available, the experiment was repeated and the
results recorded in Figure 7. To shorten the run time, the sample volume was reduced to
100 μL which also lowered the initial concentration of cells. Positive fibers were shown
to retain 98 ± 1.6% of E.coli that passed through the microfluidic channels whereas
negative fibers only retained 35 ± 1.1%. In the channels with no fibers only 53 ± 4.4% of
cells were recovered at the outlet. Cell losses could be attributed to cells adhering to the
hydrophobic channel and tubing surfaces or cells becoming trapped at the inlet or outlet
holes. Studies have shown that the isoelectric point of various bacteria range from 1.7 –
3.8 depending on the cell surface balance of carboxyl and ammonium groups and that the
isoelectric point affects adhesion to hydrophobic polymer surfaces23. A 1% BSA solution
was used to pre-coat the plastic tubing and the channel and the experiment was repeated.
Coating the channel and tubing with 1% BSA prior to experiments increased the recovery
at the outlet to 83 ±15 % of the cells. BSA has an isoelectric point at 5.4 and is negatively
charged at neutral pH24. When the tubing and channel with positive fibers was coated
with 1% BSA, any positive fibers in the channel were also saturated with BSA,
preventing E.coli from binding to the fibers during subsequent filtration. Therefore a high
number of cells at the outlet were recovered when 1% BSA was used to coat channels
with positive fibers as shown in Figure 7. Cell losses were not observed in the channels
with neutral and negative fibers possibly because the fibers fill the channel and prevent
the bacterial cells from coming in contact or settling on the channel surfaces.
120
100
CFU
80
60
40
20
0
Initial No fibers No Fibers, Negative Positive Positive
Number of
1% BSA Fibers
fibers Fibers, 1%
Cells
coating
BSA
coating
Figure 7: Nanofiber filtration of E.coli in channels with i) no fibers ii) no fibers with 1%
BSA coating iii) negative fibers iv) positive fibers v) positive fibers with 1% BSA
coating. All experiments performed in triplicate except for the positive fiber channel with
BSA coating.
4.3 Results: Epifluorescent Microscopy
While fluorescent measurements are qualitative, the benefit of this assay was that it
took only an hour to complete whereas the cell count assays on TSA plates took 2 days.
E.coli cells were stained with Syto9, a cell-permeant green fluorescent nucleic acid stain.
In order for the cells to be visible on the fibers and for the fibers to become saturated, a
high concentration of cells, approximately 7-8 logs was required. The microfluidic
devices were placed underneath the microscope and images were captured every 5
minutes using the RS Image software. In each experiment, the initial sample was injected
at a flow rate of 1 μL/min. The syringe was switched to the wash buffer at 20 minutes,
the wash buffer reached the channel at 40 minute and was allowed to run for another 20
minutes. The results of the experiments testing pH7 samples were recorded in Figure 8.
16
14
Mean Gray Value
12
10
8
6
4
2
0
0
10
20
( + ) Fibers ph4 wash
30
40
( + ) Fibers ph7 wash
50
60
70
( - ) Fibers ph7 wash
Figure 8: Graph of mean gray value over time. The initial sample is pH7 PBS buffer
spiked with approximately 107-108 CFU/mL E.coli stained with Syto9. The syringe was
switched to a wash buffer (either pH7 of pH4) at 20 min and the buffer reached the
channel at 40 min. The data shown in blue is an average of two replicates. The red and
orange data are single replicates.
As explained previously, E.coli have a net negative charge at neutral pH. In the
experiments shown in Figure 8, the negatively charged bacteria accumulated on the
positive fibers within the first few minutes. The fluorescence intensity was maintained
when the fibers (red squares) were washed with a pH7 buffer for 20 minutes. The fibers
(blue diamonds) that were washed with a pH4 buffer for 20 minutes caused the
fluorescence to drop by 84% indicating that a majority of the bacteria were eluted from
the positive fibers by washing with pH4 buffer. In the case of negative fibers (orange
circles), the negatively charged bacteria were repelled and bacteria were easily flushed
from the channel with a pH7 buffer shown by the drop in fluorescence intensity. The
fluorescent images taken at 0, 20, 40 and 60 minutes for the blue diamond data shown in
Figure 8 are shown in Figure 9.
(a)
(b)
(c)
(d)
Figure 9: Epifluorescence images corresponding to blue data shown in Figure 8.
E.coli was filtered out of a pH7 PHS buffer using positive fibers and then washed with a
pH4 buffer to elute the bacteria. The images correspond to (a) 0 minutes, (b) 20 minutes
(c) 40 minutes and (d) 60 minutes. The wash buffer reaches the channel at 40 minutes.
After the 20 minutes of washing with a pH4 buffer there is a visible reduction in
fluorescence intensity in the channel (Figure 9d vs. 9c).The fluorescent images taken at 0,
20, 40 and 60 minutes for the red square data shown in Figure 8 are shown in Figure 10.
After 20 minutes of washing with a pH7 buffer there is no noticeable change in the
fluorescence in the channel (Figure 10d vs. 10c).
(a)
(b)
(c)
(d)
Figure 10: Epifluorescence images corresponding to red data shown in Figure 8. E.coli
was filtered out of a pH7 PHS buffer using positive fibers and then washed with a pH7
buffer. The images correspond to (a) 0 minutes, (b) 20 minutes (c) 40 minutes and (d) 60
minutes. The wash buffer reaches the channel at 40 minutes.
In the second group of filtration experiments E.coli samples were prepared in pH4
buffer, filtered through negative and positive fibers and then fibers were washed with a
pH4 buffer. In these experiments, E.coli were expected to have a net positive charge
because of the low pH of the sample buffer. These filtration results were then compared
to filtration of apple juice samples spiked with E.coli (Figure 11). From the DLS analysis
of apple juice it was confirmed that there are large particles greater than 10μm in
diameter present in apple juice. For that reason, the apple juice used in the experiments
was filtered through Whatman grade 1 filter paper prior to addition of the E.coli.
E.coli in pH4 buffer (squares) attached better to negative fibers (solid squares)
producing average fluorescence intensity 1.6 times higher than the channels with positive
fibers (open squares). The results were repeated with apple juice spiked with E.coli
(triangles) producing similar results. The negative channels (solid triangles) produced
fluorescence intensity 2.0 times higher than in the positive channels (open triangles). The
higher overall fluorescence in the apple juice samples may be due to the Syto9 dye
interacting with compounds in the apple juice.
45
40
35
30
25
20
15
10
5
0
-5
0
10
20
30
40
50
ph4 buffer ( - ) fiber
ph4 buffer ( + ) fiber
Apple juice ( - ) fiber
apple juice ( + ) fiber
60
70
Figure 11: Images were analyzed in ImageJ. Mean gray value was plotted over time.
The sample buffers and wash buffer were both pH4. The syringe was switched to the
wash buffer at 20 minutes and the buffer reached the channel at 40 minutes. Apple juice
was filtered using 100 Whatman filter paper to remove any large particles. Experiments
with pH4 buffer sample filtered through negative fibers (solid square) were performed in
triplicate, pH4 buffer sample filtered through positive fibers (open square) was an
average of 2 replicates. The apple juice experiments were both single replicates.
5.0 CONCLUSION AND FUTURE WORK
The results of both the cell counting and epi fluorescence experiments show that
E.coli in a neutral pH sample can be filtered onto positively charged fibers and are
repelled by negatively charged fibers. E.coli in neutral pH samples that are filtered onto
positive fibers can be eluted by washing with a buffer with a lower pH. E.coli in pH4
buffer sample can be filtered onto negatively charged fibers and are repelled from
positively charged fibers. The samples with actual apple juice did not negatively affect
the charge interaction and filtration. However, apple juice was found to contain large
particles that could potentially clog microfluidic channels. Therefore a pre-filtration step
to remove large particles be performed.
These results show the potential for electrospun nanofibers to be used on-chip as bioseparators. Due to time constraints, some experiments in this study were not repeated in
triplicate. Further studies should be done to confirm the results. The design of the
microfluidic device can be improved by adding a second inlet hole or using a T-junction
syringe for introducing the wash buffer into the channel. This would prevent drastic
changes in pressure and backflow when the syringes are switched. If cell losses are
apparent, coating the PMMA surface with 1% BSA may be necessary. Coating the
channels by pumping 1% BSA through the device as done in this study is time
consuming. Therefore coating the PMMA surface prior to bonding is another method that
could be investigated. The quality of electrospun nanofibers can vary greatly due to small
changes in electrospinning parameters and environmental conditions. In order to design
nanofibers for filtration applications, it may be useful to determine a correlation between
spinning time and fiber pore size and fiber mat density.
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