THE QUANTIFICATION OF LIPOSOME SIGNALS
USING NANOFIBER-BASED MICROFLUIDIC
DEVICES
A Thesis
Presented to the Faculty of the Graduate School
of Cornell University
In Partial Fulfillment of the Requirements for the
Master of Engineering
by
Caitlin Elizabeth Anderson
January 2013
©2013 Caitlin Anderson
II
ABSTRACT
Microfluidic paper-based analytical diagnostics have allowed for a diversification of
analytical tools by enabling the development of inexpensive and portable devices that build upon
existing detection strategies. Taking advantage of existing quantification techniques is an
important strategy to ensure that these novel paper-based systems find use and application in the
diagnostics world. For more than 50 years high-throughput assays have been developed using
polymeric microtiter plates in which signals are quantified using specific absorbance,
fluorescence, and luminescence readers. Here, we studied the novel idea of integrating a paperbased analytical assay with a microtiter plate reader. Specifically, electrospun nanofiber mats
were designed to match dimensions and criteria of microtiter plate readers. Dye-encapsulating
liposomes were used as a model analyte and quantified using absorbance and fluorescence
detection strategies.
Initially, positively charged poly(vinyl alcohol) (PVA) and polylactic acid (PLA)
nanofibers were electrospun and functionalized in specific locations with anti-streptavidin
antibodies. Additionally, streptavidin-conjugated liposomes were synthesized to encapsulate
sulforhodamine B (SRB) (absorbance wavelength of 488 nm, and a fluorescence excitation and
emission wavelengths of 540 nm and 590 nm respectively). Liposomes were then applied and
flowed through the nanofiber mats under various conditions to investigate their selective capture,
concentration, and detection.
Primary investigations demonstrated the ability of PLA as an immobilization matrix to
selectively bind streptavidin conjugated liposomes through the use of absorbance measurements.
Fluorescence allowed subsequently for accurate readings without the interference of any of the
assay materials. The ability to specifically quantify the capture of liposomes using the microtiter
III
plate reader allowed for quantitative optimization of all involved assay steps and buffer systems
to increase the reliability of the assay. In the end, the quantification of signals was achieved with
a testing volume of 10 µL of SRB encapsulating liposomes, a wash step using 4-(2Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)-sucrose-saline buffer, and 2 µL of
detergent for liposome lysis at a concentration of 50 mM. Thus, through the use of streptavidinconjugated liposomes as a model analyte, it was demonstrated that a PLA nanofiber-based
microtiter plate could successfully detect and differentiate between different concentrations of
analytes with a detection limit of 0.5 mM and a sensitivity of 4023 Fluorescence units/mM.
IV
BIOGRAPHICAL SKETCH
Originally from Saratoga, California, Caitlin Elizabeth Anderson is the oldest daughter of
Thomas and Laura Anderson. She earned her Bachelors of Science in Biological Engineering
with a minor in Global Health from Cornell University in May of 2012. She is passionate about
the use of biological engineering to address problems of access to healthcare technologies on a
global scale. Outside of academics, she enjoys spending time with family and friends,
participating in all things athletic, and listening to music.
V
ACKNOWLEDGEMENTS
I would first like to thank Dr. Antje Baeumner, who has guided me throughout my time
in the Biological Engineering department. Her guidance has allowed me to take a chance and
pursue a career in a field that I am truly passionate about. As both a professor and an advisor,
she has been an amazing person to have the opportunity to work with.
I am so grateful to have had the ability to do research in the Bioanalytical Microsystems
and Biosensors Laboratory. I would like to thank Lauren Matlock-Colangelo who helped to
guide me throughout the development of my project. With her help I was able to successfully
maneuver my way around the lab while also accomplishing everything I could have hoped to.
Additionally, I would like to thank Sarah Reinholt for her willingness to help whenever I needed,
whether or not it was research related. I really enjoyed being able to reminisce about our
gymnastics careers in our past lives.
The Department of Biological and Environmental Engineering has been my home for the
past four and a half years, and I am thankful for all of the experiences and opportunities I was
able to have during those years. I am truly going to miss Riley Robb and the people inside of it.
Lastly, I would like to thank my family and friends who have been there to support me
from day one. It is through their support that I was able to turn my dreams into a reality.
VI
TABLE OF CONTENTS
ABSTRACT ...............................................................................................................................................III
BIOGRAPHICAL SKETCH .................................................................................................................... V
ACKNOWLEDGEMENTS ..................................................................................................................... VI
LIST OF ABBREVIATIONS .................................................................................................................. IX
INTRODUCTION..................................................................................................................................... 10
MICROFLUIDICS 2.0 ................................................................................................................................. 10
ELECTROSPUN NANOFIBERS .................................................................................................................... 11
METHODS ................................................................................................................................................ 15
MATERIALS AND CHEMICALS .................................................................................................................. 15
Materials and polymers ...................................................................................................................... 15
Buffers and Solutions .......................................................................................................................... 15
ELECTROSPINNING POLY(VINYL ALCOHOL) NANOFIBERS ...................................................................... 16
ELECTROSPINNING POLYLACTIC ACID NANOFIBERS ............................................................................... 17
LIPOSOME SYNTHESIS ............................................................................................................................. 19
NANOFIBER MICROTITER PLATE ASSEMBLY .......................................................................................... 19
Nanofiber Microtiter Plate Absorbance Image Analysis .................................................................... 20
Nanofiber Microtiter Plate Fluorescence Image Analysis ................................................................. 20
COMPARISON OF ABSORBANCE AND FLUORESCENCE READINGS ............................................................ 21
DEVICE CALIBRATION ............................................................................................................................. 21
Assay optimization .............................................................................................................................. 22
VERTICAL FLOW ASSAY .......................................................................................................................... 23
DESIGN ..................................................................................................................................................... 25
MICROFLUIDICS 2.0 DEVICE FORMATION ............................................................................................... 25
MICROTITER FLUORESCENCE READING DEVICE ...................................................................................... 27
RESULTS AND DISCUSSION ............................................................................................................... 29
INVESTIGATION OF PVA NANOFIBERS.................................................................................................... 29
INTERFERENCE OF PLA AND POREX IN ABSORBANCE ANALYSIS .......................................................... 30
Quantification of interference due to the presence of PLA and Porex ............................................... 30
Effect of PLA fiber mat thickness on microtiter plate absorbance reading ........................................ 33
ASSAY OPTIMIZATION ............................................................................................................................. 34
VII
Volume Optimization .......................................................................................................................... 34
Effect of liquid on anti-streptavidin immobilization ........................................................................... 35
Error Minimization ............................................................................................................................. 38
Liposome Lysis for Improved Signal................................................................................................... 39
CALIBRATION OF DEVICE ........................................................................................................................ 42
TIME DEPENDENCE OF ASSAY ................................................................................................................ 43
VERTICAL FLOW ASSAY ......................................................................................................................... 45
CONCLUSION ......................................................................................................................................... 47
FUTURE OUTLOOK AND STEPS........................................................................................................ 49
STRENGTHENING PVA NANOFIBERS ...................................................................................................... 49
DNA SEQUENCES AS A TARGET ANALYTE ............................................................................................. 49
REFERENCES .......................................................................................................................................... 51
VIII
LIST OF ABBREVIATIONS
DMF – Dimethylformamide
DPPC – 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DPPG – 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], sodium salt
ELISA – Enzyme-linked immunosorbent assay
HEPES – 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HSS – HEPES Sucrose Saline
MES – 2-(4-Morpholino)-Ethane Sulfonic Acid
OG – n-octyl- β-D-glucopyranoside
PBS – Phosphate buffered saline
PLA – Polylactic acid
PVA – Poly(vinyl alcohol)
SRB – Sulforhodamine B
TBS – Tris buffered saline
µPAD – Microfluidic paper-based analytical device
WHO – World Health Organization
IX
INTRODUCTION
Microfluidics 2.0
The detection and diagnosis of medical and environmental pathogens in the developing
world face challenges that prevent the use of devices and techniques that are used in more
developed areas. Inconsistent electricity, high levels of heat and humidity, and limited access to
large and expensive lab machinery limit the ability to utilize standard diagnostic technology1.
Paper-based microfluidics have the ability to overcome these challenges and detect target
analytes in resource poor settings2. The World Health Organization (WHO) has developed the
acronym ASSURED to describe the ideal characteristics of a diagnostic device of the developing
world. They specify that a device must be affordable, sensitive, specific, user friendly, rapid,
equipment free, and able to be delivered to the end user1. Microfluidic paper-based analytical
devices (µPADs) are designed to combine the abilities of traditional microfluidic devices with
the benefits of a strip-based diagnostic test2. These devices have the ability to be faster, require
smaller sample volumes, and cost less money without requiring additional external supporting
machinery or electricity. Additionally, they are capable of maintaining high levels of sensitivity
and specificity for a target analyte3. Because paper-based diagnostics rely solely on capillary
flow for fluid transport, it is possible to test a sample without the use of machinery to pump fluid
through the device. The use of hydrophobic and hydrophilic regions within a single device can
direct flow in as many ways as a traditional polymer based microfluidic device2. Because flow
occurs through the paper-based device, diffusion limitations that previously effected microfluidic
assays can be eliminated20.
10
Paper-based microfluidic devices are also highly advantageous because of their ability to
be multiplexed. Through the immobilization of different biorecognition elements, it is possible to
detect a wide range of analytes including human proteins, toxins, and even whole bacterial cells.
One technique for multiplexing µPADs is through the creation of a paper-based microtiter plate.
While µPADs are similar in function and structure to conventional microtiter plates, they provide
a cheaper and potentially easier to functionalize alternative3. An application of this technique
developed by the Whitesides lab at Harvard University is an enzyme linked immunosorbent
assay (ELISA) carried out on 96 paper-based wells4. Through such a paper-based ELISA,
diagnosis of analytes such as glucose, proteins, and enzymes can be completed in resource poor
areas that would not otherwise have access to similar diagnostic assays3,4. Quantification of a
target analyte in a given solution is extremely important for the diagnosis of medical conditions
and in monitoring of food and water supplies5. Therefore, the ability to quantify the amount of
analyte bound on a µPAD testing site has great importance. Through the use of currently existing
analyte measurement systems, it is possible to quantify the amount of analyte bound on a given
testing spot. The combination of quantification and paper microtiter plate technologies allows for
exact measurement of analyte while maintaining all of the benefits of a µPAD.
Electrospun nanofibers
Paper-based microfluidic devices are most commonly made of a cellulose membrane
onto which the biorecognition element is immobilized2. The flow of the sample solution through
the device is limited by the pore size of the membrane. In this work, we present electrospun
nanofibers as an alternative to cellulose membranes as the immobilization material for a paperbased microfluidic device. Electrospinning is a process in which a polymer in solution is spun
into a membrane of web-like fibers of diameters ranging between 1µm to 1000 nm7,19. During
11
electrospinning, the polymer spinning dope is placed inside of a syringe that is subjected to a
constant voltage. A grounded collector plate is then placed across from the syringe, and once the
attraction of the charged polymer to the grounded collector plate overcomes the effect due to
surface tension in the tip of the syringe, fibers of small diameter wick from the tip of the syringe
and land onto the collector plate. While the fibers travel from the tip of the syringe to the
collector plate, the solvent in the spinning dope evaporates leaving only the polymer behind.
Nanofibers allow for a larger surface area to volume ratio than that provided in conventional
materials, while also allowing for variability in pore size and nanofiber diameter7,8.
Electrospinning of nanofibers generates fibers that have high tensile strengths despite their small
size8. The use of nanofibers has expanded to include many applications in biological sensing.
They have been demonstrated to allow for the integration of sample preparation and
concentration into a single microfluidic system without requiring additional assay steps9.
Depending on the electrospinning procedure, it is possible to immobilize the biorecognition
element for a target analyte inside of the nanofiber prior to spinning10, 11. This would allow for a
significant reduction in the number of assay steps required for detection of the analyte.
Poly(vinyl alcohol) (PVA) and polylactic acid (PLA) nanofibers are two types of electrospun
nanofiber that can be incorporated into a biosensing device. Both PVA and PLA have the ability
to incorporate a wide variety of different particles and proteins both before and after the
electrospinning process for immobilization purposes10-112. PVA nanofibers are able to be spun
with water as a solvent, which has the potential to allow them to incorporate a larger variety of
recognition elements than PLA, which requires a much more toxic solvent12. Both PVA and PLA
nanofibers will be investigated for their ability to be incorportated into a microfluidic nanofiber
device. Scanning electron and confocal microscope images of these nanofibers shown in Figure
12
1 demonstrate the manner in which the web of electrospun nanofibers appear in the final
nanofiber mat.
30 µm
30 µm
Figure 1: Electrospun PLA nanofibers spun onto wax paper. Image A is an SEM image of PLA nanofibers using a
LEICA-440 SEM, image courtesy of Nidia Trejo, Cornell University Department of Fiber Science and Apparel
Design. Images A and B are two images taken from different regions of a nanofiber circle using the LEICA Spectral
Confocal Microscope, images courtesy of Judith Moench-Tegeder. Image B the edge of the PLA nanofiber circle,
and Image C on the bottom right depicts the center.
Positively charged nanofibers have been shown to be capable of concentrating negatively
charged liposomes out of a buffer solution9. Expanding upon this, negatively charged liposomes
will be used as a model analyte to determine the ability of nanofibers in the form of a microtiter
plate for concentration quantification. In this work, we focused on the use of Sulforhodamine B
(SRB) encapsulating liposomes that have been conjugated with streptavidin. A solution
containing these SRB encapsulating liposomes is allowed to flow through nanofibers that have
been functionalized with anti-streptavidin antibodies. The streptavidin on the surface of these
liposomes can bind to anti-streptavidin antibodies on the surface of the nanofibers, allowing for
retention of the SRB encapsulating liposomes after washing of the nanofibers. The manner in
13
which the binding of streptavitin conjugated liposomes to functionalized nanofibers is shown in
Figure 2.
Figure 2: Mechanism through which SRB encapsulating liposomes bind to hydrophilic PLA nanofibers that
have been functionalized with anti-streptavidin.
Once retained, negatively charged SRB encapsulating liposomes can be detected by changes in
absorbance at 488 nm and fluorescence at an excitation wavelength of 540 nm and an emission
wavelength of 590 nm. The fluorescence measured by the microtiter plate reader is dependent on
the concentration of SRB encapsulating liposomes that are bound to a nanofiber mat. However,
the ability of these nanofibers to effectively bind liposomes in a manner that allows for
differentiation between concentrations has yet to be determined. The ability of these nanofibers
to effectively bind SRB encapsulating liposomes as a model analyte in a quantifiable manner is
examined in this work.
14
METHODS
Materials and chemicals
Materials and polymers
Polylactic acid nanofibers created from 4043D PLA pellets from Jamplast (Manchester, MO,
USA) are the primary material used in the development of the nanofiber microtiter plate.
Nanofibers are placed on a porous POREX® membrane with bimodal pore sizes of 30 micron
pores and 8 micron pores received from Porex (Fairburn, GA, USA). Absorbant pads used are
Millipore (Billerica, Massachusetts, USA) cellulose fiber sample pads. The backing cards used in
support for the vertical flow assay were purchased from the Diagnostic Consulting Network
(Carlsbad, CA, USA). Functionalization of nanofibers was done with goat unconjugated antistreptavidin purchased from Vector Laboratories (Burlingame, CA, USA). Strepdavidin
conjugated liposomes were made with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and
1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], sodium salt (DPPG) bought from
Avanti Polar Lipids (Alabaster, AL, USA). The sulforhodamine B (SRB) encapsulated inside
these liposomes was purchased from Molecular Probes Incorporated (Eugene, Oregon, USA).
Absorbance readings were taken using the BioTek PowerWave XS Microplate
Spectrophotometer and fluorescence readings were taken using the Biotek FLX 800 Microplate
Fluorescence Reader (Winooski, VT, USA).
Buffers and Solutions
All buffers used were made of chemicals purchased from the following vendors; Fischer Biotech
(Wembley WA, Australia), J.T. Baker (Philipsburg, NJ, USA), Macron Fine Chemicals
(Philipsburg, NJ, USA), EMD (Darmstadt, Germany), and MP Biomedicals (Santa Ana, CA,
15
USA). Each buffer solution was prepared as a stock solution from which desired concentrations
were created through dilution with Millipore water. These stock solutions include 10X PBS, 10X
TBS, 10X HSS buffer, 1% w/r Casein, 0.4 M Sodium Carbonate, 5% Tween 20, and 10%
Sodium azide.
Electrospinning Poly(vinyl alcohol) nanofibers
PVA nanofibers were spun using hexadimethrine bromide modified PVA to have a positive
charge at pH 7. A solution consisting of 0.4 g of PVA and 3 g of deionized (DI) water were
placed in a small vile. The vile was then placed in the gravity oven for four hours at 95C,
allowing for the PVA to dissolve completely in the water. While the PVA in DI water cooled,
0.04 g of polybrene and 1 g of DI water were added to another small glass vile. The polybrene
solution was then allowed to dissolve in water. The polybrene dissolved in water could then be
added to the PVA solution. After the combination of the two, 0.06 g of triton X was added to the
polymer solution. The polymer spinning solution was then vortexed on high for two minutes
until well mixed. The spinning solution could then be poured into a 5 ml plastic syringe with a
20 gauge needle. While resting horizontally to allow the solution to settle, aluminum foil was
wrapped around the copper collector plate and placed into the cardboard setup. The syringe was
placed in the syringe pump set with the following parameters; volume set to 3 ml and a flow rate
of 0.01 ml/min. The cardboard setup was placed into the center of the plastic spinning box, with
the collector set to be 15 cm from the tip of the spinning needle. The grounding wire was
connected to the back of the collector plate, and the high voltage power voltage power source
wire was connected to the tip of the spinning needle. The voltage was turned on to 15 kV. For
each sample, the syringe pump and voltage source ran for 120 minutes total.
16
Figure 3: Spinning device apparatus used for the spinning of PVA nanofibers. All electrospinning is done inside of a
grounded fume hood.
The manner in which each of the components is connected during the electrospinning processs of
PVA nanofibers is shown in Figure 3.
Electrospinning Polylactic Acid nanofibers
Polylactic acid (PLA) nanofibers were created and spun at 22wt% in the Frey Lab at Cornell
University. Polylactic acid solution was created by placement of 1.35 g PLA pellets and 5 mL
Dimethylformamide (DMF) placed into a glass vial with a stir bar. The vial was then placed on
the hot plate at about 70 ºC and a stir setting of 8. The polymer was heated for 1.5 hours, with a
vortexing step halfway through preventing the stir bar from getting stuck in the solution. After
the PLA pellets fully dissolve into a homogeneous solution, the spinning solution was placed in a
17
5 mL glass syringe with a needle gauge of 20. The flow rate on the syringe pump was set to 10
µL/min, the voltage was set to 15 kV, and the distance from the needle tip to the collector plate
was set to 10 cm. The syringe was allowed to sit for 10 minutes in the syringe holder in order to
bring the solution up to temperature prior to spinning. Figure 4 shows how each of these
components for electrospinning are assembled to allow for effective fiber mat production.
Figure 4: Spinning device apparatus used for the spinning of PVA nanofibers. Procedure is completed inside of a
grounded fume hood.
The heating element in which the syringe sits was set to a temperature of 70 ºC. The heat gun
was set to 70 ºC +/- 5 ºC and aimed to heat the polymer inside the needle. Samples were spun
onto wax paper that had been secured onto a copper plate using electrical tape. Rotations of 90
degrees occurred every 4 minutes, allowing a total of 16 minutes to spin each PLA nanofiber
sample
18
Liposome Synthesis
Liposomes were synthesized using the standard protocol created by Dr. Katie A. Edwards15-17.
All liposomes created contained 150 mM SRB. SRB was chosen for its colorimetric and
fluorescent properties. In terms of fluorescence, SRB can be detected by an excitation
wavelength of 540 nm and an emission wavelength of 590 nm. Synthesis of SRB encapsulating
liposomes utilized the reverse phase evaporation method and separated by size using extrusion
and size exclusion chromatography15. Liposomes were subsequently characterized using a
Bartlett Assay to determine phospholipid content in each sample16. Liposomes were tagged with
COOH on their surface to allow for conjugation with streptavidin.
Nanofiber microtiter plate assembly
Creation of the nanofiber microtiter plate starts with the creation of the immobilization surface
on which testing is to take place. PLA and PVA nanofibers were investigated for use as
immobilization surfaces. All nanofibers were spun onto wax paper to allow for ease of removal
after electrospinning is complete. Using an 5/16” McGill hole punch, nanofibers are cut into
approximately 8 mm circles while attached to the wax paper onto which they were spun. Using
tweezers, each circle was able to be easily removed from the wax paper. Nanofiber circles were
then stored on a flat surface surrounded on top and bottom by aluminum foil to stabilize the
fibers prior to functionalization.
19
Nanofiber Microtiter Plate Absorbance Image Analysis
Absorbance measurement of PLA nanofiber samples after testing with streptavidin liposomes
was completed by digital imaging of pictures taken of each sample immediately after testing.
Absorbance levels were measured using ImageJ and analyzing the absorbance values over each
circle with relation to a negative control.
Nanofiber Microtiter Plate Fluorescence Image Analysis
Fluorescence measurement of samples occurred using the Biotek FLX 800 Microplate
Fluorescence Reader. PLA nanofiber samples were placed into the reading device that allowed
for support and consistency between readings. The sample and reading device combination were
placed into the microtiter plate reader and aligned accordingly. Fluorescence readings of
streptavidin liposomes occurred at an excitation wavelength of 540 nm and an emission
wavelength of 590 nm. For each of these readings it was found to be ideal to read at and intensity
of 40.
Quantification of interference in the microtiter plate reader using dye
Food coloring was used in order to quantify the interference due to the presence of PLA and
porex in the microtiter plate reader. One 50 µL drop of food coloring was added to 1000 µL of
DI water in a small glass beaker. 50 µL of the well-mixed solution was added to each of 5 wells
in a single column. The 5 rows represented the following: (1) solution only, (2) solution pipetted
on top of porex, (3) solution pipetted on top of PLA which is subsequently flipped, (4) PLA and
porex with solution pipetted onto the side facing down, and (5) porex placed on top of solution.
Another 1000 µL of DI water was added to the small glass beaker. The procedure was repeated
20
as described above for 5 wells in each row, with concentration decreasing from left to right. In
the six well, 50 µL of DI water was placed. The seventh well contained just the material in
question, which meant that it was either empty, contained PLA or porex, or both. The plate was
read in the plate reader at an absorbance of 490 nm by both point and area scan to quantify the
interference of these materials in the absorbance readings output from the microtiter plate reader.
Comparison of Absorbance and Fluorescence readings
Fluorescence and absorbance readings were taken of PLA nanofiber circles tested with
streptavidin liposome concentrations of 0, 0.25, 0.5, 1, 1.5, 2.5, and 3.5 mM. Fluorescence
readings were taken using the Biotek FLX 800 Microplate Fluorescence Reader with an
excitation wavelength of 540 nm and an emission wavelength of 590 nm. Absorbance readings
were taken by measuring the total color intensity across a nanofiber circle using ImageJ
software. The two were compared graphically using Matlab through the creation of a best-fit
curve using least squares data fitting.
Device calibration
Samples of PLA spun onto wax paper in the Frey lab on were cut into circles of an 8 mm
diameter using a McGill hole punch. Circles were isolated onto non-laminated Porex in a square
formation with 1 mm between one another. Samples were placed in a dish containing prewetting
solution until the solution had fully soaked through. Pre-wetting solution consisted of 1 mL each
of methanol, 10x TBS, and Millipore water. The solution was changed after every 5 circles to
minimize differences due to the order in which the circles were selected. The samples were then
21
dried for 1.5 hours in the vacuum oven at 40 °C and 15 Hg. The PLA circular fiber mats then
received 10 µL of anti-streptavidin solution via hand spotting. Anti-streptavidin hand spotting
solution consists of 1 part anti-streptavidin antibodies dissolved in 0.1M 2-(4-morpholino)ethane sulfonic acid (MES) for every 1 part 0.4 M NaHCO3/Na2CO3 with a pH of 9. Samples
were then dried for 1.5 hours in the vacuum oven at 40 °C and 15 Hg. Each sample was then
blocked on each hydrophilic spot by pipetting 10 µL of streptavidin liposome assay blocking
solution directly onto the Porex and the dried overnight in the vacuum oven at room temperature
and 15 in. Hg. The stav liposome assay blocking solution consisted of 0.1% Tween-20, 0.1%
Na-Casein, and 0.25% Sucrose. The fiber mat and Porex combination was then transferred to sit
on top of a piece of absorbant pad. Samples were tested horizontally by pipetetting 10 µL of
different concentrations of streptavidin liposomes directly onto each circular fiber mat. They
were then washed with 1x HEPES saline sucrose (HSS) buffer pipetted directly onto each spot.
Each spot was washed with 20 µL of buffer three times. In every case, this procedure was
repeated at least three times.
Assay optimization
Analyzing effect of dissolving medium for anti-streptavidin
The effect of dissolving medium for anti-streptavidin was measured by analyzing fluorescence
data for five liposome concentrations for each case. Prior experiments had been carried out with
anti-streptavidin dissolved in 500 µL of Milipore water. The results from the previous standard
were compared to nanofibers functionalized with anti-streptavidin dissolved in 500 µL 0.1 M
MES. In both cases, liposome concentrations of 0.25, 0.75, 1, 1.25, and 2 mM were used for each
22
of the three data sets. Readings were taken immediately using the fluorescence microtiter plate
reader at the previously specified settings.
Liposome lysis prior to fluorescence reading
An additional lysis step was added to the previously described assay protocol to allow for an
increase in measured fluorescent signal. Liposomes were lysed using n-octyl- β-Dglucopyranoside (OG). The effect of volume on OG was investigated first to find the ideal
volume at which lysed liposomes are not washed from the fiber mat. Starting with a maximum10
µL of solution, 5 volumes were used in increments of 2 µL on tested PLA nanofiber circles each
containing 10 µL of 1 mM streptavidin liposomes. . Measurements were taken in the
fluorescence microtiter plate reader before and after the liposome lysis step to allow for
measurement of only the effect of OG to each sample. Six concentrations of OG were then tested
by diluting 60 OG to test at 10, 20, 30, 40, 50 and 60 mM OG prior to application on PLA
nanofiber circles containing bound streptavidin liposomes. Each circle was immobilized with
anti-streptavidin and tested with 10 µL of 1 mM of streptavidin liposomes. These concentrations
were tested using 2 µL of OG, the volume that had been previously determined to be ideal for
signal amplification. Again, fluorescence readings were taken before and after application of the
respective OG solution.
23
Vertical Flow Assay
Samples of PLA spun onto wax paper in the Frey lab were cut into circles of an 8 mm diameter
using a McGill hole punch. Circles were then isolated onto non laminated Porex in a rectangular
formation with 1 mm between one another. The fiber mat and Porex combination was then
placed on top of an absorbant pad to facilitate flow through the fibers. Hydrophilic regions were
then created on the samples on the left column by pipetting 20 µL of pre-wetting solution. Prewetting solution consisted of 1 mL each of methanol, 10x TBS, and Millipore water. Each
nanofiber circle intended to be made hydrophilic was allowed to soak in prewetting solution
before being returned to the Porex sheet. The samples were then dried for 1.5 hours in the
vacuum oven at 40° C and 15 in. Hg. After removal from the oven, samples were attached to an
absorbant pad using a backing card as shown in Figure 5.
Absorbant Pad
PLA Nanofibers
Backing Card
Porous Porex
Figure 5: Pictorial representation of the vertical assay assembly consisting of PLA nanofibers, Porex, an absorbant
pad, and a backing card. .
The fully assembled assay was then placed in solution containing SRB and DI water with only
the tip of the Porex exposed to solution in order to track the movement of liquid through the
device. The assay was allowed to run for three minutes, until liquid had passed through the entire
device. The assay was then transferred to a solution consisting of 1xHSS buffer to allow for the
food coloring to be rinsed from the nanofibers. Fluorescence readings were taken after the wash
step.
24
DESIGN
Microfluidics 2.0 Device Formation
Current nanofiber-based diagnostics assays using SRB encapsulating streptavidin
liposomes give a colorimetric qualitative response*CITATION*. It is possible to utilize existing
fluorescence and absorbance microtiter plate readers for the quantification of liposome
concentration bound to these diagnostic assays. To do so, a nanofiber diagnostic must be
designed in a manner that allows for accurate readings produced from a microtiter plate reader.
The American National Standards Institute has defined the dimensions of a microtiter plate to
consist of 96 individual wells in the form of eight rows and twelve columns. The diameter of
each well is standardized at 8 mm, with a 1 mm spacing between each and 5 mm from the
outside wells to the edge of the plate18. In order to receive accurate readings, a nanofiber
microtiter plate must follow the aforementioned dimensions. Each “well” in the nanofiber
microtiter plate will consist of an 8 mm nanofiber circle. These circles can be created through the
use of an 8mm hole punch on a nanofiber mat spun onto wax paper. Once they have been cut,
PLA nanofiber circles can be easily removed from the wax paper and arranged on a piece of
porous porex in the following arrangement. The completed nanofiber microtiter plate with the
appropriate dimensions is shown in Figure 6.
25
Figure 6: Microtiter plate consisting of electrospun nanofibers. The base of the plate is made of porex, which has the
ability to absorb the solution when it is washed off the test locations. The spacing of each circle is important to allow
for this absorption as well. In our case, we are going to allow for 1 mm spacing between each well. The space
between the outer circles and the edge of the plate will be 5 mm. The size of a plate will therefore depend on the
number of analytes which are intended to be tested for. In the case above, with 96 total wells, the total size of the
plate would be 8.1 by 11.9 cm.
In the formation of the microtiter plate, circles can be cut very closely to one another on the
nanofiber mat. Leaving a 5 mm border around the entire mat to allow for support during creation
of each circle, a total of 80 nanofiber circles can be generated from a 10 cm by 10 cm nanofiber
mat. Each of these circles is then individually placed onto a piece of porous porex upon which
the application of each step of the assay takes place. During each of these steps, an absorbant pad
will be placed underneath the porex layer to facilitate flow through the nanofibers and to absorb
any liquid that flows through. After testing has been completed, the absorbent pad must then be
removed in order to allow for precise and accurate readings from the microtiter plate reader. The
reader shines light from the bottom of the “plate”, therefore it is important that there is not a
26
visual obstruction between the light and the sample. The complete arrangement of the porex,
nanofibers, and absorbant pad are depicted in Figure 7.
Figure 7: Graphical representation of the nanofiber, absorbent pad, and porex setup. The absorbent pad, as described
before, is removed prior to imaging in the microtiter plate reader.
To start out, testing will occur by pipetting the solution directly onto each dot. The volume
required to saturate the entire dot is unknown, and would need to be determined. Once the
appropriate volume has been determined, immobilization of anti-streptavidin must be optimized
to maximize signal while minimizing error. Additionally, overall assay modification will have to
be examined to determine the most effective way to generate signal from a nanofiber microtiter
plate. The most important assay modification that will be examined is the addition of a step to
lyse streptavidin liposomes bound to each nanofiber circle. Through the optimization of the
nanofiber microtiter plate assay, a calibration curve for streptavidin liposomes can be developed.
Microtiter fluorescence reading device
The fluorescence microtiter plate reader is arranged to take readings of a microtiter plate at a
given location inside of its assembly. Accurate readings for the nanofiber microtiter plate are
reliant on the placement of each nanofiber circle inside of the device. To ensure appropriate
placement, it was necessary to develop a reading device inside which the nanofiber microtiter
plate sits. This device was developed according to the specifications for microtiter plates as
27
defined by the American National Standards Institute. The reading device created consists of two
main parts. The first of these is a wood support frame designed to fit into the microtiter plate
reader. A wood ridge on the inside of the frame was placed for appropriate support of the
nanofiber microtiter plate. In between the nanofiber microtiter plate and the wood support frame,
a thin plastic sheet is placed to prevent contamination of the frame and of the reader. The type of
plastic used must be chosen to prevent interference due to auto-fluorescence. The manner in
which each of these pieces fit together is depicted below.
Figure 8: Assembly of microtiter fluorescence reading device for placement in the microtiter plate reader. Wood
support frame is 8.1 by 11.9 cm with slots into which the nanofiber microtiter plate rests.
After completion of the assay with streptavidin liposomes, the nanofiber based microtiter plate
can be placed on top of this device and placed inside of the Biotek FLX 800 Microplate
Fluorescence Reader. The device is designed to allow for consistent placement of the respective
circles so that fluorescent readings among one and between multiple nanofiber microtiter plates
are accurate and precise. Through the combination of the nanofiber microtiter plate and its
corresponding reading device, it is possible to utilize the technology of a microtiter plate reader
for the quantification of fluorescence in a nanofiber sample.
28
RESULTS AND DISCUSSION
Investigation of PVA fibers
Initially, poly(vinyl alcohol) nanofibers were investigated due to their biocompatibility and
simplicity in electrospinning. Negatively charged nanofibers of a variety of thicknesses were
generated and tested on their ability to withstand fluid flow to allow for use in the nanofiber
microtiter plate. PVA nanofibers were found to require structural support in order to support
wicking through the mat at the volumes desired. Imaging of the wicking of colored liquid
allowed for a better understanding of what was happening to the nanofibers as liquid was
flowing through them.
10 nm
Figure 9: Negatively charged PVA nanofiber mat after wicking experiment with red food coloring. Data courtesy of
Judith Moench-Tegeder.
During the wicking experiment, it became obvious that the PVA nanofibers were unable to
maintain their structure after exposure to an aqueous solution. Figure 9 depicts a segment of
nanofiber through which only some was exposed to the wicking solution. On the upper right
hand corner, where the red food coloring is the most dense, the nanofiber structures are unable to
29
be seen. In the orange region of the mat, with food coloring at non-saturating levels, the
nanofiber structures are starting to lose their shape. Only in the lower right hand region of the
fiber mat, where no solution has yet reached, can the fibrous structure of the PVA nanofibers be
seen. The inability of the PVA nanofibers to withstand the force of aqueous flow required that a
more stable nanofiber be determined for use in the nanofiber microtiter plate. Polylactic acid was
chosen because of its ability to be functionalized with anti-streptavidin liposomes and to
withstand lateral flow through the fibers.
Interference of PLA and Porex in Absorbance analysis
In development of the nanofiber microtiter plate, the effect of the materials used, i.e. PLA
nanofibers and Porex support, was examined with respect to the subsequent quantification of
captured liposomes. Understanding the influence of these materials on device readings allow for
maximization of the signal to noise ratio for the nanofiber microtiter plate.
Quantification of interference due to the presence of PLA and Porex
The absorbance in the BioTek Powewave XS microtiter plate reader is determined by measuring
the amount of light of a specific wavelength that passes through the sample. For this reason, it
was necessary to investigate the ability of PLA and Porex to absorb light at our wavelength of
interest. The greater the ability of these materials to absorb at this wavelength, the greater it
interferes with he liposome reading. The interference of PLA and Porex in the microtiter plate
absorbance readings was determined by analyzing red dye of known concentrations with various
combinations of the two materials. The presence of PLA and Porex was found to have a
significant effect on the absorbance reading obtained from the plate reader from these
measurements. The data from the full area scan is shown in Figure 10. It demonstrates how the
30
presence of these materials incrementally increases the absorbance reading for the same
concentration of dye.
5
3
1
0
Dye only
Dye and Porex
Dye and PLA
Dye, Porex and PLA
Figure 10: Full area scan of each of 4 different cases with incrementally decreasing concentrations of dye as read on
the microtiter plate reader at an absorbance of 490 nm. The higher the absorbance, the more red there is in each of
the samples. As absorbance decreases, the samples become more blue in color.
In the full area scan, it is visible that the presence of PLA and Porex has a significant impact on
the absorbance reading from the microtiter plate. Comparing these samples to the one containing
dye only, the Porex and PLA are shown to cause an increase in the absorbance reading of each
well. This signifies that the two materials are scattering light at the wavelength of interest. The
last column in Figure 10 shows each case with just DI water. The presence of Porex and PLA is
shown to lead to a significant increase in absorbance reading. Interestingly, the samples in the
last column, which contain no liquid with each sample, show higher absorbance interference than
the similar case with just water. This is important to note, because it will affect further studies
completed using this technique. It will be necessary to image the samples in the plate reader
31
when they are still wet because it allows for a reduction in interference due to Porex and PLA.
The end point scan provided similar results, while also providing numerical data of the
absorbance readings as they related to the known concentration of dye in each well. The graph in
Figure 11 depicts the absorbance values for the control, PLA, and Porex and PLA together along
a range of concentrations.
Effects of PLA and Porex on absorbance readings
4.5
4
Absorbance (OD)
3.5
3
2.5
Liquid only
2
PLA
1.5
Porex and PLA
1
0.5
0
0
0.01
0.02
0.03
0.04
Concentration (µl dye/ µl water)
0.05
Figure 11: Absorbance readings for known concentrations of dye as read by a microtiter plate reader for three cases;
liquid only, PLA, and Porex and PLA.
The control wells and those containing PLA both appeared to follow fairly linear curves,
demonstrating an even effect over various concentrations and samples. It is obvious, however,
that there is definite interference as the absorbance readings at a concentration of 0 µL/µL differ
by about 2 OD. The error between absorbance readings for nanofibers exposed to the same
concentration of dye may was higher than the variability for wells containing only liquid. The
32
interference of PLA nanofibers in the absorbance readings may be dependent on differences in
thicknesses between different nanofiber samples.
Effect of PLA fiber mat thickness on microtiter plate absorbance reading
The thickness of each PLA fiber mat was thought to play a role in the ability of those fibers to
influence the absorbance readings. In order to determine the effect of the thickness of each fiber
mat on the absorbance reading, the thicknesses of 20 PLA nanofiber circles were measured using
the confocal microscope. They were then measured in the absorbance microtiter plate reader at a
wavelength of 490 nm. The relationship between nanofiber thickness and its interference in the
absorbance reading is represented in figure 12.
Effect of PLA Nanofibers on Absorbance Readings
2.5
Absorbance (OD)
2
1.5
1
0.5
0
0
10
20
30
40
Fibermat Thickness (µm)
50
60
Figure 12: Known thicknesses as measured by the confocal microscope were placed into the microtiter plate reader
and the absorbance was read at 490 nm. The fibers were measured in the confocal at a gain of 575 and offset of -3%.
33
The data from the microtiter plate reader shows that the absorbance readings of PLA fiber mat
remain fairly constant as thickness increases. The absorbance readings are demonstrated to
arrange around an optical density value of 2 independent of fiber mat thickness. This could be
used as a baseline from which to measure various dye concentrations, however because the
reader is only capable of reading up to 4 OD it decreases the range of possible readings by a
factor of two. For nanofiber mats of similar thicknesses, the absorbance reading is not identical
which reduces the ability to use an initial reading of the fiber mat as a baseline from which
further measurements can be taken. The fact that PLA nanofiber mats are not consistent in
thickness across 8 mm, let alone the area of an entire mat, removes the ability to subtract
potential noise due to this interference. Based on these measurements, it was determined that
fluorescence readings of streptavidin liposomes bound to PLA nanofibers would be the most
accurate method of detection. Streptavidin liposomes fluoresce with an excitation wavelength of
540 nm and an emission wavelength of 590 nm, conditions under which PLA nanofibers and
Porex do not provide any form of interference. The switch to fluorescence reading for detection
of streptavidin liposome binding has the potential to increase the overall signal to noise ratio.
Assay optimization
Volume optimization
Preliminary testing with PLA nanofiber circles demonstrated that the original volumes used in
the assay were insufficient to provide consistent results. The first set of tests was done through
the application of each solution during the functionalization process through pipetting. After
streptavidin liposomes were applied to the functionalized nanofibers, it was visible that fluid
flow occurred only in specific spots on each fiber mat. As fluid flows through a fiber mat, it
34
chooses the path of least resistance, which means that it will flow through the thinnest part of a
given fiber mat. Even though each mat is relatively the same thickness, there are variations up to
50% on the scale of a single 8 mm circle. Upon close examination, it was determined that
prewetting was the limiting step in the functionalization process. After this realization, the
prewetting step was modified to allow the PLA nanofibers to soak in the solution until all regions
of the fiber mat became hydrophilic before moving on to the next step. This modification was
successful, and subsequent testing with streptavidin liposomes demonstrated that flow was
occurring through the entire fiber mat and no loner in select points. Optimization of the volumes
of liquid (other than the prewetting solution) pipetted onto each nanofiber circle also was
necessary to maximize signal while minimizing amount of each solution required. For the steps
requiring minimization of solution volume, 10 µL was determined to be sufficient to allow for
flow through the entire area of the fiber mat. For the washing steps, full removal of all unbound
streptavidin liposomes is required. Therefore, three washing steps of 20 µL of buffer were found
to successfully wash the nanofiber mats.
Effect of liquid on anti-streptavidin immobilization
For lateral flow assays using anti-streptavidin, the anti-streptavidin used is dissolved in 500 µL
of 0.1M MES to yield a 2 µg/µL final concentration. However, testing with PLA nanofibers is
done with anti-streptavidin dissolved in 500 µL of DI water to also yield a final concentration of
2 µg/µL. The effect of using one dissolving liquid over the other was tested to determine which
would give more accurate results. The same assay was run using nanofibers of the same fiber
mat functionalized with the two types of anti-streptavidin. From this test, it was found that one
set of data was more consistent than the other based on the variation within data points of the
same liposome concentration.
35
The effect of liposome concentration on fluorescence for anti-streptavidin dissolved in
deionized water is shown in Figure 14. Previous testing with streptavidin-conjugated liposomes
on nanofibers was completed with DI water as the dissolving medium, therefore it was initially
anticipated to provide consistent results.
Calibration Curve for Anti-streptavidin in Water
4000
3500
Fluorescence
3000
2500
2000
1500
1000
500
0
0
0.5
1
1.5
2
2.5
Concentration (mM)
Figure 14: Graphical representation of fluorescence data for varying concentrations of streptavidin liposomes from
the microtiter plate reader. Three data points taken for each concentration point are demonstrated by their average
and standard deviation in this graph for PLA nanofibers functionalized with anti-streptavidin dissolved in water and
tested with 10 µL each.
In this case, anti-streptavidin dissolved in DI water was shown to have deviations that varied so
significantly that little information could realistically be retrieved. In strictly colorimetric tests
with PLA nanofibers, 1 µL of streptavidin liposomes is used successfully to generate a visible
signal. For the development of a device that can detect a wide range of concentrations of analyte,
the inconsistency between different concentrations pose a serious problem in the accuracy and
precision of the device. The results for anti-streptavidin dissolved in 0.1 M MES were found to
be slightly more consistent, as depicted in Figure 15.
36
Calibration Curve for Anti-streptavidin in 0.1M MES
3000
Fluorescence
2500
2000
1500
1000
500
0
0
0.5
1
1.5
2
2.5
Concentration (mM)
Figure 15: Graphical representation of fluorescence data from the microtiter plate reader. Three data points taken
for each concentration point are demonstrated by their average and standard deviation in this graph for PLA
nanofibers functionalized with anti-streptavidin dissolved in 0.1M MES and tested with 10 µL each.
The results from anti-streptavidin dissolved in 0.1M MES were found to have large variations at
a given concentration of streptavidin liposomes as well. However, it also could be noted that the
variations in data from this second set was found to be more consistent over the range of
concentrations. The value of the standard deviation for each of the five data points in the second
set of data was found have an average of 551 fluorescence units (FUs) and standard deviation of
130. Based on this data, it was determined that it would be most beneficial to use antistreptavidin in 0.1M MES for all concentration assays from this point forward. An investigation
of techniques to minimize the error at each data point was required to generate an accurate and
reliable concentration curve for the streptavidin liposome assay.
37
Error Minimization
In all previous experiments, inconsistencies between data points that lead to a high level of error
made it difficult to generate a precise concentration curve. Through modification of the liposome
assay, it was possible to minimize these deviations. The modifications consisted of adjusting the
volumes used, changing the anti-streptavidin used, and utilizing nanofiber circles of similar
thicknesses. Six concentrations were analyzed to determine how changes in the amount of
streptavidin liposomes used the fluorescent reading for the sample (Figure 16).
Fluorescence Reading based on Liposome
Concentration
8000
7000
Fluorescence
6000
5000
4000
3000
2000
1000
0
0
0.5
1
1.5
2
2.5
Concentration (mM)
3
3.5
4
Figure 16: Graphical representation of fluorescence data from the microtiter plate reader. Eight data points taken for
each concentration point are demonstrated by their average and standard error in this graph for PLA nanofibers
functionalized with Anti-streptavidin in 0.1 M MES and tested with 10 µL of streptavidin conjugated liposomes.
The general trend demonstrates that as the concentration of liposome increases, the fluorescence
reading also increases. At the lower concentrations, the difference is not large enough to be able
38
to significantly differentiate between them. Even though there is a greater increase between
fluorescent readings at the higher concentrations, the error accompanied by these points also
makes it difficult to significantly differentiate between them.
Liposome Lysis for Improved Signal
The effect of quenching of the fluorescent signal when the SRB is encapsulated within a
liposome may limit the maximum fluorescent signal read for a given concentration. Lysing of
liposomes immediately before measuring the fluorescent signal may allow for these closely
concentrated SRB molecules to spread out and increase the total signal at each concentration.
Through the addition of a lysing step, it became possible to more significantly differentiate
between fluorescent readings at each liposome concentration (Figure 17).
Effect on fluoresence signal by liposme lysis
20000
18000
16000
Fluorescence
14000
12000
10000
Pre Lyse Step
8000
Post Lyse Step
6000
4000
2000
0
0.25
0.5
1
1.5
Concetration (mM)
2.5
3.5
Figure 17: Graphical representation of fluorescence data from the microtiter plate reader before and after the lysis of
streptavidin liposomes. Three data points taken for each concentration point are represented by their average and
standard error in this graph for PLA nanofibers functionalized with anti-streptavidin dissolved in 0.1M MES which
were each tested with 10 µL of liposomes.
39
The results from this experiment show that the inclusion of a lysis step allowed for a significant
increase in the fluorescence reading.
In the two highest concentrations, the increase in
fluorescence was found to be the greatest. This increase was expected, as the lysis of liposomes
allows the SRB inside of each liposome to be detected without the effect of dye quenching. At
the lower concentrations the increase was found to be less significant. At 0.25 mM, however, a
decrease in the fluorescence reading was found. This may have been due to the fact that the large
volume of OG used lead to a portion of the lysed liposomes to be washed away. The decrease
would only have been seen in the smaller concentrations because of the dramatic increase in
fluorescence reading that the lysis of the liposomes causes. It is also important to note how the
lysis step affected the measured standard deviation for each point. At the lower concentrations,
there was little change in the deviation from the mean. However, at the higher concentrations the
deviation was increased significantly. This deviation may be affected by the concentration and
volume of OG used for the lysis of streptavidin liposomes.
Effect of volume on signal after liposomes lysis
The volume of OG used for lysis of SRB encapsulating liposomes was evaluated to determine its
effect on the final fluorescence reading. During preliminary investigations of a liposome lysis
step it was seen that larger volumes of OG appeared to wash away some of the SRB that had
been released from the liposomes. Additionally, a volume of OG that is too small could
potentially be unable to reach all liposomes in a given fiber mat. Therefore, a range of OG
volumes were used to determine which would be the most effective for the lysis of liposomes.
40
Effect of OG volume on signal amplification
Fluorescence Increase (%)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
2
4
6
Volume OG (µL)
8
10
12
Figure 18: Varying volumes of 30 mM OG were used for the lysis of 1 mM of streptavidin liposomes immobilized
on PLA nanofiber circles. Fluorescence readings were taken using an excitation wavelength of 540 nm and an
emission wavelength of 590 nm. The percentage of fluorescence increase was calculated by dividing the difference
between before and after readings by the post-lysing fluorescence reading.
As previously expected, the greater the volume of OG solution used on a PLA nanofiber circle
containing bound streptavidin liposomes, the more likely it was for a fraction of SRB to be
washed away. The greatest signal increase was found with a volume of 2 µL of OG. In addition,
the variability between the data points from that volume was found to be small enough for the
increase to be significant.
Effect of concentration on signal after liposome lysis
Finalization of the nanofiber microtiter plate assay required a better understanding of the effect
of the concentration of OG used to lyse the streptavidin liposomes. This was done by evaluating
the fluorescence increase of PLA nanofiber circles with 10 µL of 1 nM of streptavidin liposomes
for six concentrations of OG all tested at the volume determined to be the most effective
previously (Figure 19).
41
Effect of OG concentration on signal amplification
Fluorescence Increase (%)
1
0.95
0.9
0.85
0.8
0.75
0.7
0.65
0.6
0.55
0.5
0
10
20
30
40
Concentration OG (mM)
50
60
70
Figure 19: Varying concentrations of 2 µL of OG were used for the lysis of 1 mM of streptavidin liposomes
immobilized on PLA nanofiber circles. Fluorescence readings were taken using an excitation wavelength of 540 nm
and an emission wavelength of 590 nm. The percentage of fluorescence increase was calculated by dividing the
difference between before and after readings by the post-lysing fluorescence reading.
The effect of OG concentration on total signal amplification was found to be less significant than
the effect of the volume used. The range between all six concentrations was found to be between
84 and 94%. The maximum values were found at 50 and 60 mM OG. Because these values were
so similar, and the error for both cases was found to be fairly low, a final OG concentration of 50
mM was determined to be the most effective for further liposome assays in the nanofiber
microtiter plate.
Calibration of device
The results from each of the assay optimization steps were taken into account in the
determination of a final calibration curve for the detection of streptavidin liposomes using a PLA
nanofiber microtiter plate (Figure 20).
42
Calibration Curve for PLA Nanofiber Microtiter Plate
35000
30000
Fluorescence
25000
20000
15000
10000
5000
0
0
0.5
1
1.5
2
2.5
Liposome Concentration (mM)
3
3.5
4
Figure 20:Calibration curve for the PLA nanofiber microtiter plate tested with different concentrations of
streptavidin liposomes. Each fiber mat was functionalized with anti-streptavidin dissolved in 0.1 M MES prior to
testing and lysed with 2 µL of 50 mM OG after exposure to streptavidin liposomes. Eight data points were taken for
each concentration. Given this data, a limit of detection of 0.5 mM and a sensitivity of 4023 FUs/mM were
calculated.
The introduction of a liposome lysis step provided for greater differences in fluorescence for
varying concentrations of liposomes. Additionally, there was a decrease in the variability for
each individual concentration because of the addition of an appropriate lysis step. Based on this
calibration curve it is possible to determine the concentration of streptavidin liposomes by
application to the PLA nanofiber microtiter plate and measurement in the fluorescence microtiter
plate reader.
Time dependence of assay
All of the fluorescence readings up until this point have been taken immediately after assay
completion. The effect of time on the fluorescence reading was investigated by measuring 5
43
samples at each concentration over the course of a week. In between each measurement the
device was stored wrapped in aluminum foil and kept in a moisture controlled container to
prevent contamination. The results from these measurements are shown in Figure 21.
Time dependence on Fluoresence Readings
25000
Fluorescence
20000
15000
Day 1
Day 2
Day 4
10000
Day 5
Day 7
5000
0
0.25
0.5
1
1.5
Concentration (mM)
2.5
3.5
Figure 21: Graphical representation of the time dependence of fluorescence readings from the microtiter plate
reader. Each concentration was measured at 5 times over the course of 7 days and are demonstrated in this graph
along with their measured standard deviations.
These data demonstrate a similar trend to what was seen during the addition of a liposome lysis
step. Over time, the fluorescence reading of each sample is found to increase. The largest
increase is found to be between day one and day two, after which the change in signal appears to
approach an asymptote. The increase is due to the lysis of drying liposomes and consequential
release of the entrapped SRB dye. The increase in signal over time also is shown to lead to an
increase in the difference between each concentration. This may allow for a more sensitive assay
that can better differentiate between differences in concentration. With respect to the deviation of
44
each concentration, it is seen that with an increase in time, there is an increase in the variability
for these samples. This increase was seen much more drastically at the higher concentrations
than the lower ones. This is similar to what was demonstrated with the addition of OG to each
sample. Therefore, for the improvement of the assay it will be ideal to utilize this increase in
signal, most likely through the use of OG, because it will allow for greater control and ideally
more consistent results.
Vertical Flow Assay
It is important to note that for each data set the standard deviation for the negative control is
significantly smaller than any of the other data points. This could suggest that the fluorescence
reading, which is dependent on the amount of binding for each PLA nanofiber circle, is also
dependent on the area available for binding in each. During the functionalization of each circle,
the area through which the volume flows varies slightly between each circle. This is due to the
thickness of the circles, which is not 100% consistent between circles from the same nanofiber
mat. The absorption of solution through a smaller area could potentially be addressed by soaking
the circles in each solution, and not just the pre-wetting solution. If this is the case, it would
encourage the use of a lateral or vertical flow device consisting of these circles that would allow
for exposure to each solution in the entire PLA nanofiber circle and not just the areas where it
absorbs through the most quickly. The development of a vertical flow assay depended on the
ability of selectively hydrophilic nanofiber circles to pull liquid through them. Initial
investigations used red dye to evaluate the ability of these circles to do so. They demonstrated
successful flow through hydrophilic nanofiber circles; however quantitative data was unable to
be retrieved. Therefore, it is possible to use this methodology with a solution containing
liposomes to generate quantitative results. Ten circles were exposed to prewetting solution
45
allowing them to become hydrophilic. Another three circles acted as the negative control, and
were kept hydrophobic. Upon exposure to a solution containing SRB-encapsulating liposomes,
liquid wicked upwards through the nanofiber mats and eventually into an absorbant pad helping
to facilitate flow. The lack of flow through hydrophobic nanofibers could be visibly seen by the
naked eye. Quantitative data was retrieved that also supported this observation (Figure 22).
Ability of hydrophilic PLA nanofibers to absorb in a vertical
flow assay
4000
3500
Fluorescence
3000
2500
Hydrophobic
2000
Hydrophilic
1500
1000
500
0
PLA Nanofiber Samples
Figure 22: Analysis of the ability of hydrophilic nanofiber circles to absorb liquid in a vertical flow assay through
the use of 2 mM SRB encapsulating liposomes. Fluorescence was measured with an excitation wavelength of 540
nm and an emission wavelength of 590 nm for ten PLA nanofiber circles.
The ability of circular PLA nanofibers to vertically absorb liquid allows for them to be used in a
vertical flow assay configuration. The large increase between the two data sets demonstrates that
the fibers that have been exposed to prewetting solution are highly capable of drawing and
collecting liposomes in solution. This proves promising for the expansion of future uses for PLA
nanofiber microtiter plates for analyte detection. In addition to the benefits of soaking a PLA
nanofiber mat in a given solution, a vertical flow assay format would allow for mass testing
without requiring the use of micropipettes for exact measurement.
46
CONCLUSION
Initial investigations with PVA nanofibers found they had difficulties maintaining their
delicate structure under the force of a liquid falling onto them. Therefore, PLA nanofibers were
determined to be ideal for the development of a microtiter nanofiber plate. Streptavidin tagged
liposomes were able to be retained and quantified with functionalized electrospun PLA
nanofibers. Variation between nanofibers from the same fiber mat lead to inconsistencies in
initial data received. However, optimization of the assay by examination of each step in the
process allowed for minimization of possible error regardless of the properties of each individual
nanofiber circle. It was determined that the use of anti-streptavidin dissolved in 0.1 M MES
allowed for more consistent data than when dissolved in water.
Primary results using streptavidin liposomes demonstrated a steadily increasing
fluorescence signal with increase in concentration of streptavidin liposomes, however large
deviations at each concentration made it difficult to differentiate between one another. The
addition of a liposome lysis step allowed for greater separation of fluorescence data for different
concentrations by allowing encapsulated SRB to be released, therefore reducing the effect of
quenching of the fluorescent molecules. Further investigation on the ability of the nanofiber
microtiter plate to detect streptavidin liposomes would need to take place to generate a consistent
assay for the detection of streptavidin liposomes.
The possibility for high throughput testing of an analyte was determined to be feasible
through the use of a vertical flow assay. The introduction of a vertical flow assay provides the
ability to reduce the inconsistencies in flow due to thickness variations between 8 mm nanofiber
mat circles. The ability of this technology to be used in high throughput testing would be
improved by adjusting the manner in which each nanofiber circle is generated. Difficulties
47
associated with manipulation of 8 mm circles could be avoided by generating hydrophilic
nanofiber circles in a nanofiber mat that makes up the entire nanofiber plate. Melting wax or
another hydrophobic material onto all regions not to be tested would generate the nanofiber
microtiter plate. The simplification of the nanofiber microtiter plate could allow for an increase
in the number of applications for this technology.
The usefulness of a nanofiber microtiter plate is limited by the ability to minimize effects
due to variation between nanofiber circles. Depending on the analyte used, this variation may
play a significantly greater role than what was demonstrated in this work. For each analyte, assay
optimization would be required to allow for the greatest possible signal to noise ratio. The
variation in thickness between nanofiber mats could potentially be reduced through the
modification of the electrospinning process. The ability of electrospun nanofibers, even when
inconsistent, to generate a consistent signal for different concentrations of SRB-encapsulating
liposomes demonstrates their ability to be a beneficial immobilization surface in diagnostic
testing. Future applications for a nanofiber microtiter plate in the developing world range from
detection of enteric diseases in humans to Listeria in food and agriculture, as it allows for
quantitative detection of the analyte without the need for large machinery.
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FUTURE OUTLOOK AND STEPS
Strengthening PVA nanofibers
Exposure of PVA nanofibers to an aqueous solution demonstrated that the fibers were unable to
maintain their structure under those conditions. The addition of an adhesive layer for support
provided some structure, however results were not consistent enough to be able to utilize PVA
nanofibers in a nanofiber microtiter plate. The development of a technique to strengthen these
nanofibers that would allow them to be exposed to solution without losing their form would
expand the range of uses for the nanofiber microtiter plate. As a biocompatible nanofiber, PVA
can be used for a variety of uses inside and outside of the body. In addition, it does not require as
sophisticated of conditions during the electrospinning process as PLA. Therefore, the
development of a strengthening method, whether it be physical or chemical, that allows for
nanofiber stabilization on the range of an 8mm diameter would allow for expansion of the
current technique to a number of new applications.
DNA sequences as a target analyte
Expansion of the applications of a nanofiber microtiter plate to include the detection of analytes
that play a more significant role in healthcare, food processing, and environmental safety would
allow for a greater societal impact. The structure of nanofibers may facilitate the
functionalization of target DNA probes onto their surface. The use of DNA sequences as a target
analyte would require further knowledge about the surface chemistry of PLA nanofibers to allow
for the development of a functionalization method that is effectively able to immobilize DNA
probes on the surface. The use of another nanofiber polymer, such as PVA may allow for the
immobilization of DNA probes inside of the nanofibers themselves. The use of very strong
49
solvents in development of PLA nanofibers prevents the immobilization of DNA probes during
the electrospinning process. PVA, on the other hand, does not require strong solvents in their
creation. Therefore, it could be possible to directly spin DNA probes with PVA to generate a
functionalized nanofiber. This would eliminate the need for additional functionalization of
nanofibers prior to testing with the target analyte.
50
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