CHEMICAL MODIFICATION OF POLYSTYRENE AND GOLD SURFACES
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
Alexander David Roth
August 2009
© 2009 Alexander David Roth
ABSTRACT
Investigations toward the surface modification of gold and polymers and the long-term
storage of functionalized surfaces were carried out. First gold was modified with
cystamine to see if amine functional groups could be formed on gold surfaces. Data
were inconclusive as an acid orange 7 assay determined low amine group coverage but
Schiff’s reagent (fuscin-sulfurous acid) test showed a subsequently higher aldehyde
group coverage when coupling glutaraldehyde to the aminated surface. Second, biotin
and Ru(bpy3)2+ were conjugated using NHS-ester and amine conjugation chemistry,
however, no coupling was observed. Third, carboxylic acid groups were generated on
polystyrene (PS) and polymethyl methacrylate (PMMA) using UV and ozone
treatment and quantified using water contact angle to measure hydrophobicity, and
utilizing a toluidine blue O assay for the carboxylic acid surface concentration. Longterm storage under various storage conditions including temperature, buffer
components and pH were tested. An initial water contact angle of 42.0o and
carboxylic acid group density of 12.4 nmol/cm2 were obtained for PS. In general, even
under best conditions, an initial loss of functionality was observed reaching a steady
state condition after 1 hour of storage. It was also determined that the temperature of
storage did not affect hydrophobicity or carboxyl surface concentration. Samples
stored in dry conditions maintained a significantly higher surface concentration than
samples stored in 0.1 M phosphate buffer. PMMA retained 100 % surface
functionality after heating up to 90oC, 95oC, and 100oC, while becoming more
hydrophobic, indicating the loss of functional groups that are not carboxylic acid.
Conversely, heated polystyrene did not become hydrophobic, but lost about 40% of its
carboxyl surface groups. Samples coated with acetyl acetone were very hydrophobic,
and carboxyl surface concentration was inconsistent. Hydrophobicity tended to
increase with pH for HEPES, Tris, and sodium borate buffer, with water and Tris
maintaining fairly hydrophilic surfaces (52.5-55.9o). However, the best carboxyl
surface concentration was achieved on pH 5 and 7 water and pH 5 PBS. Thus, the best
conditions for storage are dry stored conditions, acidic and neutral water, or acidic
PBS buffer.
ii
BIOGRAPHICAL SKETCH
Alexander David Roth graduated from Oceanside High School in June of 2005.
Following this, he went to pursue a B.S. in Biological and Environmental Engineering
at Cornell University. Alex completed his B.S. by January 2009, and went on to do his
Master’s of Engineering in Agricultural and Biological Engineering at Cornell
University. While at Cornell, Alex was treasurer of the Cornell Chapter of Institute of
Biological Engineers and participated as a peer advisor for the College of Engineering.
Additionally, Alex served as a teaching assistant for bioengineering kinetics and
thermodynamics course for two semesters.
In the summer of 2007, Alex performed research at Cornell Weill Medical College in
New York City under the direction of Dr. Baolin Wang of the Department of Genetic
Medicine. There, Alex performed research on analyzing the sonic hedgehog (Shh)
pathway, and how mutations in the pathway can cause neurological and skeletal
mutations in mice and people. In the summer of 2008, Alex performed research at
Roswell Park Cancer Institute under the direction of Dr. Daryl Nazareth in the
department of Medical Physics. Alex’s research involved optimization of radiation
treatment applied to prostate cancer patients using intensity modulated radiation
therapy.
Alex is currently performing research under the direction of Dr. Antje J. Baeumner in
the Department of Biological and Environmental Engineering at Cornell University. In
his research, Alex analyzed surface modification of gold and polystyrene, and how
these modifications can be effected based on thermal and chemical treatments, and
how storage conditions may also affect surface treatment.
3
To my parents, Jeffrey and Ronnie Roth, my first real teachers.
4
ACKNOWLEDGMENTS
This work was done with the help of the National Institute of Health (NIH) and the
Cornell Nanobiotechnology Center (NBTC). Major contributors to the success of this
research include Antje J. Baeumner, Sam R. Nugen, and Peter J. Asiello.
5
TABLE OF CONTENTS
Chapter 1: Functionalization of Gold Plated Surfaces
12
1.1 Introduction
12
1.2 Materials and Methods
13
1.2.1
Amination of Gold Surface
13
1.2.2
Acid Orange 7 (AO7) Assay for Quantification of
13
Amine Surface Concentration
1.2.3
Glutaraldehyde Conjugation on Aminated Gold
14
Surfaces
1.3 Results and Discussion
15
1.4 Conclusions and Future Work
16
Chapter 2: Electrochemiluminescence
20
2.1 Introduction
20
2.2 Materials and Methods
22
2.2.1 Conjugation of Biotin and Ruthenium
22
2.2.2 ECL Readings Using Streptavidin-Coated Beads
22
2.3 Results and Discussion
23
2.4 Conclusions and Future Work
25
Chapter 3: Long-Term Stability of Hydrophobicity and Carboxyl
29
Functionality on Polystyrene Surfaces
3.1 Introduction
29
3.1.1 Polystyrene: Design of a Microfluidic Device
29
3.1.2 Stability of UV and Ozone Treated Surfaces
29
6
31
3.2 Materials and Methods
3.2.1 Sample Preparation
31
3.2.2 Water Contact Angle Analysis
31
3.2.3 Toluidine Blue O (TBO) Assay for Quantifying
32
Carboxyl Surface Concentration
3.3 Results and Discussion
33
3.3.1 Analysis of Controls
33
3.3.2 Forty Eight Hours after Initial Treatment
35
3.3.3 Seven Days after Initial Treatment
37
3.3.4 Four Weeks after Initial Treatment
39
3.4 Conclusions and Future Work
41
Chapter 4: The Affects of Heating, Surface Coating, and Buffer Storage
47
on Hydrophobicity and Carboxyl Functionality of UV-Treated Polystyrene
and PMMA Surfaces
4.1 Introduction
47
4.1.1 PMMA: Design of a Microfluidic Device
47
4.1.2 Thermal and Chemical Effects on Carboxyl Surface
48
Chemistry and Hydrophobicity
4.2 Materials and Methods
50
4.2.1 Sample Preparation
50
4.2.2 Water Contact Angle Analysis
51
4.2.3 Toluidine Blue O (TBO) Assay for Quantifying
51
Carboxyl Surface Concentration
4.3 Results and Discussion
51
4.3.1 PMMA and Polystyrene: Thermal Bonding
55
4.3.2 Polystyrene and Solvent Bonding
55
7
4.3.3 Buffer and pH Experiments
4.4 Conclusions and Future Work
56
58
8
LIST OF FIGURES
Figure 1.1: AO7 Calibration for quantifying amine surface concentration
15
Figure 1.2: Quantification of amine surface concentration on gold plates
16
Figure 2.1: Desired conjugation of NHS-PEG4-Biotin with
23
Ru(bpy)2(phen-5-NH2)(PF6)2
Figure 2.2: Conjugation product of biotin and ruthenium in the presence of
24
DMSO
Figure 2.3: ECL detection levels of pure streptavidin, biotin and
25
ruthenium, and streptavidin bound to biotin and biotin-ruthenium
conjugate product
Figure 3.1: Calibration curve for determination of carboxyl surface
34
concentration on UV treated polystyrene surfaces
Figure 3.2: Average water contact angles for non-stored polystyrene
35
Figure 3.3: Average carboxylic acid surface concentration for non-stored
36
polystyrene
Figure 3.4: Average water contact angle for polystyrene 1 hr, 5 hr, 24 hr,
36
and 48 hr after UV and ozone treatment
Figure 3.5: Average carboxyl surface concentration for polystyrene 1 hr, 5
37
hr, 24 hr, and 48 hr after UV and ozone treatment
Figure 3.6: Average water contact angle for polystyrene 24 hr, 48 hr, 4
38
days, and 7 days after UV and ozone treatment
Figure 3.7: Average carboxyl surface concentration for polystyrene 24 hr,
39
48 hr, 4 days, and 7 days after UV and ozone treatment
Figure 3.8: Average water contact angle for polystyrene 7 days, 14 days, 3
weeks, and 4 weeks after UV and ozone treatment
9
40
Figure 3.9: Average carboxyl surface concentration for polystyrene 7
40
days, 14 days, 3 weeks, and 4 weeks after UV and ozone treatment
Figure 4.1: Water contact angle analysis for PMMA control samples
52
Figure 4.2: Water contact angle analysis for UV and Ozone treated
53
PMMA and polystyrene heated samples
Figure 4.3: Carboxylic acid surface quantification for PMMA control
54
samples
Figure 4.4: Carboxylic acid surface quantification for UV and Ozone
55
treated PMMA and polystyrene heated samples
Figure 4.5: Water contact angle analysis for polystyrene with a surface
56
coating of acetyl acetone
Figure 4.6: Carboxylic acid surface quantification for polystyrene with a
57
surface coating of acetyl acetone
Figure 4.7: Water contact angle analysis for UV and ozone treated
58
polystyrene stored in various solutions for 1 hour at either acidic,
neutral or basic pH
Figure 4.8: Carboxylic acid surface quantification for UV and ozone
treated polystyrene stored in various solutions for 1 hour at either
acidic, neutral or basic pH
10
59
LIST OF TABLES
Table 3.1: Average water contact angles for UV and ozone treated
43
polystyrene samples stored over a four week duration
Table 3.2: Average carboxyl surface concentration for UV and ozone
44
treated polystyrene samples stored over a four week duration
Table 4.1: Average water contact angles on the surface of heated and UV
61
and ozone treated polystyrene and PMMA
Table 4.2: Average carboxylic acid surface concentration on the surface of
61
heated and UV and ozone treated polystyrene and PMMA
Table 4.3: Average Water Contact Angles for UV and Ozone Treated
62
Polystyrene samples stored in various buffers at acidic, neutral, or
basic pH
Table 4.4: Average carboxylic acid surface concentration for UV and
Ozone Treated Polystyrene samples stored in various buffers at
acidic, neutral, or basic pH
11
62
CHAPTER 1
Functionalization of Gold-Plated Surface
1.1: Introduction
Thiols are often used for functionalizing gold surfaces, facilitating the immobilization
of biomolecules onto gold1. However, amine modification is interesting because it can
utilize the disulfide bond for gold attachment while expanding the kinds of molecules
that can be immobilized onto gold surfaces. Cystamine (C4H12N2S2) and cysteamine
(C2H7NS) are compounds that utilize a disulfide bond for attachment of amine
groups2. This is important as amines are often used as a base for the attachment of
other molecules3. Cystamine has been used in the past to immobilize many oxidationreduction sensitive proteins2. N-Hydrosuccinimide (NHS) esters are often conjugated
to amine groups as a way to covalently couple proteins onto surfaces4. Additionally,
amines can be easily conjugated with most protein functional groups and peptides
groups, especially those with carboxylic acid functional groups4.
In addition to amine conjugation with carboxylic acids being useful toward
immobilization of proteins, amines can conjugate with other amines via linker
molecules. Aldehyde and amine conjugation chemistry is well studied, and
glutaraldehyde makes a good linker between amine functional groups5 and also in the
development of polymers6. Additionally, the use of glutaraldehyde as a linker has
allowed for use of immobilization of amino acids, peptides, and proteins7,8. This is
important for the surface immobilization of detection proteins such as streptavidin
(which will be discussed more in the following section and in chapter 2).
12
In the experiments described, amine was functionalized onto gold surfaces using
cystamine as the conjugation solvent. Following cystamine conjugation, aldehyde
groups were conjugated to the amine surfaces and qualitatively analyzed to determine
if aldehyde conjugation was successful. An acid orange 7 (AO7) assay was used to
quantify the amine functionalization on the gold surface and compared to amine
quantification using similar protocols.
1.2: Materials and Methods
1.2.1: Amination of Gold Surface
Gold surfaces were functionalized according to a protocol used by Wirde and Gelius2.
Before functionalization, all gold slides were cleaned by soaking 1 hr in 98% ethanol.
Samples were washed with deionized water and dried with N2 following the ethanol
soak. This was followed by exposing the gold surfaces to 254 nm UV light using a
Jelight UVO-Cleaner, model# 144AX. The machine had a power of 28 mW/cm2 while
the light source emitted light at approximately 1cm away from the surface. Samples
were exposed for fifteen minutes followed by a two minute flush with air. Gold slides
were soaked in a 2.5 mM cystamine in water solution for twenty-four hours.
Following functionalization, cystamine slides were rinsed with ethanol and dried with
N2.
1.2.2: Acid Orange 7 (AO7) Assay for Quantification of Amine Surface
Concentration
13
Gold slides were immersed in a 1.0 mM AO7 solution in pH 12 water. The gold slides
remained immersed in the solution for three hours at room temperature. After the
immersion, the gold slides were rinsed three times with pH 12 water and left to dry.
After the slides were dried, the acid orange 7 dye was desorbed by placing the gold
surfaces on pH 3 water and shaking for 15 minutes. Absorbance was measured using a
spectrophotometer. A standard curve was created for 100 µM, 10 µM, 5 µM, 1 µM,
500 nM, and 100 nM concentrations of AO7. Absorbance was measured at a
wavelength of 460 nm. Surface concentration was calculated using the following
equation:
(1)
A = p*c
Where A is the absorbance, c is the concentration, and p is a constant. A sample
calibration of an AO7 assay can be seen in figure 1.1
1.2.3: Glutaraldehyde Conjugation on Aminated Gold Surfaces
The amine-gold surface was subsequently coupled to glutaraldehyde. A protocol for
conjugation was used similar to that in the bioconjugate techniques book12. Gold slides
were immersed in 0.1 M pH 9.5 sodium borate (NaBO3) buffer. Glutaraldehyde was
added to make a 1:1 molar ratio of aldehyde to amine functional groups. Sodium
cyanoborohydride (NaCNBH3) was added to form a 0.05M solution of NaCNBH3 in
sodium borate buffer. The reaction was allowed to proceed for two hours at room
temperature. Slides were removed from solution and rinsed with deionized water and
dried with N2. 1 mL of Schiff’s reagent was applied to each surface to determine the
14
Acid Orange 7 Calibration
y = 15327x
R2 = 0.9998
Absorbance at 460 nm
10
1
0.1
0.01
0.001
0.0000001
0.000001
0.00001
0.0001
AO7 Concentration (M)
Figure 1.1 AO7 Calibration for quantifying amine surface concentration. Concentration of amines
is equal to concentration of dye. Surface concentration can be calculated by multiplying the dye
concentration by the desorbing volume and dividing by the area of desorption.
strength of aldehyde functionalization on the gold surface. The color change observed
determined the concentration of aldehydes present on the gold surface.
1.3: Results and Discussion
Ultimately, amination of the gold surface was successful using cystamine. However,
the maximum surface concentration was not attained with these experiments (see
figure 1.2). While amine surface chemistry did increase when gold was in the presence
of cystamine, the value 0.145 nmol/cm2 is less than the 1 nmol/cm2 obtained by other
groups2. This conjugation concentration was even low compared to the total
concentration of amines on gold after 1 hour of a cystamine in gold soak2.
Nonetheless, the low surface concentration may not be a problem. If large protein
molecules bond to the surface amine groups on gold, intermolecular forces may allow
proteins to spread out over the electrode surface.
15
Surface Concentration (nmol/cm^2)
Amine Surface Chemistry on Gold
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
No Cystamine
Cystamine
Treatment
Figure 1.2 Quantification of amine surface concentration on gold plates.
Schiff’s Reagent should turn magenta or purple in the presence of a high concentration
of aldehydes10. The application of Schiff’s reagent to surfaces plated with cystamine
and glutaraldehyde caused a dark blue color to be immediately produced. This
indicates a very strong presence of aldehydes (though the quantification of the amount
of aldehydes is unknown). This may mean that that amount of desorbed acid orange 7
could have underestimated the amine surface concentration on the gold slides.
1.4: Conclusions and Future Work
Amination of the gold surface was successful using 2.5 mM cystamine in water.
However, the results of the AO7 assay also indicate that the maximum surface
concentration of amines was not reached2. The results of the Schiff’s reagent test
indicate a strong presence of aldehydes on the gold surface. The result of the very
successful conjugation between the aminated gold surface and glutaraldehyde
16
indicates that the acid orange 7 assay may have underestimated the total surface
concentration of amine functional groups. A future test might be to get a different read
on the amine surface chemistry on gold using a different test.
It is also possible that if the amine concentration calculated by the AO7 assay is
accurate, the amine surface concentration may be sufficient to immobilize proteins or
peptides onto the gold surface. Intermolecular forces and steric effects will limit the
amount of amines that can conjugate with proteins. Using glutaraldehyde as a linker
molecule will also have a minor impact on the total concentration of protein that can
bind to the gold surface.
17
REFERENCES
1. Hostetler, M.J., Stokes, J.J., Murray, R.W. Infrared Spectroscopy of ThreeDimensional Self-Assembled Monolayers: N-Alkanethiolate Monolayers on
Gold Cluster Compounds. Langmuir, 12, 1996. pp. 3604-3612.
2. Wirde, M., Gelius, U. Self Assembled Monolayers of Cystamine and
Cysteamine on Gold Studied by XPS and Voltammetry. Langmuir, 15, 1999.
pp. 6370-6378.
3. Doron, A., Katz, E., Willner, I. Organization of Au Colloids as Monolayer
Films onto ITO Glass Surfaces: Applications to the Metal Colloid Films as
Base Interfaces to Construct Redox-Active Monolayers. Langmuir, 11, 1995.
pp. 1313-1317.
4. Abello, N., Kerstjen, H.A.M., Postma, D. Selective Acylation of Primary
Amines in Peptides and Proteins. Journal of Proteome Research, 6, 2007. pp.
4770-4776.
5. Chang, M.C., Tanaka, J. FT-IR Study for Hydroxyapatite/Collagen
Nanocomposite Cross-linked by Glutaraldehyde. Biomaterials, 23, 2002. pp.
4811-4818.
6. Hardy, P.M., Nicholls, A.C., Rydon, H.N. Nature of Glutaraldehyde in
Aqueous Solution. Chemical Communications, 1969. 565-566.
7. Hopwood, D., Allen, C.R., McCabe, M. The Reactions Between
Glutaraldehyde and Various Proteins; An Investigation of Their Kinetics.
Histochemical Journal, 2, 1970. pp. 137-150.
8. Hopwood, D. Theoretical and Practical Aspects of Glutaraldehyde Fixation.
Histochemical Journal, 4, 1972. pp. 267-303.
9. Hermanson, G.T. Bioconjugate
10. Techniques; 2nd Edition. 2008. pp. 265-268.
18
11. Bourne, G.H., Danielli, J.F. International Review of Cytology: The
Chemistry of Schiff’s Reagent. 10, 1961. pp. 1-93.
19
CHAPTER 2
Electrochemiluminescence
2.1: Introduction
ECL is the application of voltage in the presence of a luminescent molecule, which
causes the emission of light1. During an ECL reaction, ruthenium complex usually in
the form of tris(bipyridine) ruthenium (II) (Ru(bpy3)2+) emits a photon of light at a
wavelength of 620 nm. This emission is caused by the excitation after ruthenium is
reduced by tripropylamine (TPA) during the application of an electric potential. TPA
and ruthenium are oxidized on the electrode. TPA then loses a proton, forming TPA
radical, the form of TPA that ultimately reduces ruthenium to its excited state.
Since the state of ruthenium is heavily tied to the presence of an applied potential,
ECL has become popular for detection and modeling systems. Ruthenium does not
require a light source or radiation for excitation, and it can be used for fairly low levels
and a wide range of detection2. Additionally, ruthenium has been used in the detection
of amine functional groups, with stronger reactions occurring in the presence of
tertiary amines3. However, a stronger detection can be obtained by functionalizing the
ruthenium protecting group with other functional groups. This is necessary as many
biological molecules lack tertiary amine groups for necessary detection4.
Often, Ru(bpy3)2+ will be produced with a NHS ester functionalized onto the
protecting group of ruthenium5, as NHS-ester and amine bonding is well studied6. The
NHS-ester linkage allows for ruthenium to be conjugated directly onto amino acids,
20
allowing it to be used as a protein label7. Ruthenium may also be produced with other
functional groups to conjugate with other molecules that are useful for detection
studies. One molecule that ruthenium has been conjugated to is PEG-biotin. Biotin has
a very strong binding affinity for streptavidin, with a KD or dissociation constant of
around 10-15 M8. In addition, biotin may have high affinity for other proteins (such as
avidin), though streptavidin is most often used for binding with biotin.
The process of modifying a molecule by conjugation with biotin (known as
biotinylation) has been used for peptide and protein modification and detection9.
Streptavidin and biotin binding chemistry can even hold well in the presence of strong
detergents, which could otherwise damage cellular proteins or cause the lysis of a cell
or liposome10. This strong binding chemistry between biotin and streptavidin under
cellular lytic conditions can mean efficient purification of biotinylated molecules11.
While this may pose a problem for further protein purification, it is very convenient
for detection purposes, which rely on the strong binding affinity for measurement of
sensitivity.
In this experiment, ruthenium was conjugated to biotin utilizing standard NHS-ester
and amine conjugation reactions. Four biotin molecules can bind to one streptavidin
molecule12. The coupling of streptavidin with a surface protein and biotin with
ruthenium allows for long term detection of ECL. Following a biotin-ruthenium
conjugation, ECL measurements were taken, comparing the level of ECL between
streptavidin, the biotin-ruthenium conjugation, and the biotin-ruthenium conjugate
bound to the streptavidin.
21
2.2: Materials and Methods
2.2.1: Conjugation of Biotin and Ruthenium
NHS-PEG4-biotin was conjugated to bis(2,2’-bipyridine)-(5aminophenanthroline)ruthenium bis (hexafluorophosphate) (Ru(bpy)2(phen-5NH2)(PF6)2), using NHS-ester and amine binding chemistry6. The ruthenium was
diluted to 11.1 mM in PBS pH 7.2. Biotin was added to a concentration of 6.8 mM.
The mixture was lightly shaken for two hours at room temperature. The conjugate was
stored at -20oC for further studies. A second conjugation test was used, dissolving
dimethyl sulfoxide (DMSO) to a concentration of 2.1mM in the PBS before adding the
ruthenium. This was done to assist in the dissolving of the ruthenium complex in
solution (see figure 2.1).
2.2.2: ECL Readings using Streptavidin-Coated Beads
To determine the effectiveness of the conjugation, ECL readings were taken of the
conjugates in the presence of Dynal Myone T1 streptavidin beads. Beads were
separated out of solution with magnets for one to two minutes, with the solution being
removed after separation. Beads were resuspended in PBS pH 7.2 to the same volume.
The beads were separated out, and the PBS buffer was removed. This was repeated for
a total of three PBS washes. Following the last wash, the biotin-ruthenium conjugate
in solution was added to the beads. Conjugates were diluted to a concentration of 400
nM. The conjugates and the beads were gently shaken for 15 minutes at room
temperature. After shaking, the beads and attached conjugates were separated from the
supernatant using magnets. The supernatant was discarded, and the beads were
resuspended in B+W buffer, which contained 5 mM Tris and HCl, 0.5 mM
22
ethylenediaminetetraacetic acid (EDTA), and 1 M NaCl at pH 7.5. This was repeated
for a total of five washes. Samples were diluted 1/60 in a solution containing PBS,
TPA, and TritonX-100 for ECL readings. ECL readings were measured using an
Organon Teknika Nuclisens Reader. Samples were flushed with cleaning buffer
containing KOH and TritonX between readings. Samples analyzed include a “blank”
sample, streptavidin beads independent of biotin and ruthenium, conjugates by
themselves, and bead-conjugate couples. All ECL readings were outputted to a
computer. The strength of the reading is directly linear to the total concentration of
ruthenium present in the solution.
2.3: Results and Discussion
During the second conjugation experiment, the ruthenium was removed from its
protecting group by application of the DMSO (see figure 2.2). Thus, while the
+
NHS-PEG4-biotin
Ru(bpy)2(phen-5-NH2)(PF6)2
PBS Buffer, pH 7.2
2.5 hour shake at room temp
Biotin-Ruthenium Conjugate
Figure 2.1 Desired conjugation of NHS-PEG4-Biotin with Ru(bpy)2(phen-5-NH2)(PF6)2.
23
conjugation might have been successful, ECL would not have been detected
Results of the ECL test show that ruthenium was not fully conjugated to biotin (see
figure 2.3). If conjugation was successful, ECL readings for conjugated samples with
streptavidin should have been much greater than that of pure ruthenium conjugate.
However, the results show that some of the conjugation was successful because the
average ECL value of the ruthenium conjugate in the presence of biotin was still about
five times greater than that of pure streptavidin.
Figure 2.2 Conjugation product of biotin and ruthenium in the presence of DMSO. The clear
orange layer represents the PBS and DMSO solution where the reaction was performed. The opaque
orange layer is the biotin conjugated to the protecting group of ruthenium. At the bottom is a small
black layer, which is ruthenium without the protecting group
24
ECL Readings
2500
ECL Value
2000
1500
1000
500
0
Streptavidin
Conjugate
Streptavidin + Conjugate
Sample Contents
Figure 2.3: ECL detection levels of pure streptavidin, biotin and ruthenium, and streptavidin
bound to biotin and biotin-ruthenium conjugate product.
2.4: Conclusions and Future Work
Since NHS-ester and amine surface chemistry is well studied, the likely event is that
the conjugation did not occur as a result of other factors affecting the experiment.
While DMSO did dissolve ruthenium more quickly into PBS, it coincided with the
separation of the metal from the protecting group, allowing conjugation to occur, but
(most likely) without ECL. A better way to facilitate the reaction would be to use a
buffer that would dissolve ruthenium more easily without affecting the protecting
group. Any buffer can be used so long as it does not contain amines (such as tris), as a
buffer with amines would compete with the ruthenium protecting group for
conjugation with biotin. Additionally, if biotin were exposed to too much air, the
moisture could have dissolved it, preventing any reactions from occurring with biotin.
Minimizing moisture exposure to biotin before the reaction would allow for effective
biotin-ruthenium conjugate to form to be used in further ECL studies.
25
Once the conjugation is successful, the biotin ruthenium conjugation can be used as a
label for the desired molecule of detection. One such way to use biotin and ruthenium
for targeting cellular compounds is to encapsulate the conjugate into liposomes, which
can permeate cell membranes much more easily than pure conjugates13. Additionally,
liposomes can be used as models of cell membranes13,14. Biotin-ruthenium conjugates
labeled with DNA or RNA can bind to the DNA or RNA of a desired sample sequence
to detect the presence or absence of a gene in an organism, or even an organism
itself15. Future work is going to involve encapsulation of the conjugate into liposomes,
and labeling with DNA and mRNA of organisms for detection purposes.
26
REFERENCES
1. Pyati, R., Richter, M.M. ECL-Electrochemical Luminescence. Annu. Rep.
Prog. Chem. Section C, 103, 2007. pp. 12-78.
2. Blackburn, G.F., Shah, H.P., Kenton J.H., et. al. Rapid
Electrochemiluminescence Assays of Polymerase Chain Reaction Products.
Clinical Chemistry, 37, 1991. pp. 1626-1632.
3. Knight, A.W., Greenway, G.M. Relationship Between Structural Attributes
and Observed Electrogenerated Chemiluminescence (ECL) Activity of Tertiary
Amines as Potential Analytes for the tris(2,2’-bipyridyl) ruthenium (II) ECL
Reaction. Analyst, 121, 1996. pp. 101R-106R.
4. Fang, L., Lü, Z., Wei, H., et. al. Quantitative Electrochemiluminescence
Detection of Proteins: Avidin-Based Sensor and tris(2,2’-bipyridine)
ruthenium (II) Label. Biosensors and Bioelectronics, 23, 2008. pp. 1645-1651.
5. Miao, W., Bard, A.J. Electrogenerated Chemiluminescence. 72. Determination
of Immobilized DNA and C-Reactive Protein on Au (111) Electrodes Using
Tris(2,2’-bipyridyl) Ruthenium (II) labels. Analytical Chemistry, 75, 2003. pp.
5825-5834.
6. Bragg, P.D., Hou, C. Subunit Composition, Function, and Spatial
Arrangement, in Ca2+-Activated and Mg2+-Activated Adenosine
Triphosphatases of Escherichia coli and Salmonella typhimurium. Arch.
Biochem. Biophys., 167, 1975. pp. 311-321.
7. Staffilani, M., Hǒss, E., Giesen, U., et. al. Multimetallic Ruthenium(II)
Complexes as Electrochemiluminescent Labels. Inorganic Chemistry, 42, 2003.
pp. 7789-7798.
27
8. Slim, M., Sleiman, H.F. Ruthenium (II)-Phenanthroline-Biotin Complexes:
Synthesis and Luminescence Enhancement Upon Binding to Avidin.
Bioconjugate Chemistry, 15, 2004. pp. 949-953.
9. Lassman, M.E., Kulagina, N., Taitt, C.R. Fragmentation of Biotinylated Cyclic
Peptides. Rapid Communications in Mass Spectrometry, 18, 2004. pp. 12771285.
10. Roesli, C. Neri, D., Ryback, J. In vivo Protein biotinylation and Sample
Preparation for the Proteomic Identification of Organ- and Disease- Specific
Antigens Accessible from the Vasculature. Natural Protocols, 1, 2006. pp. 192199.
11. Rybak, J.N. Scheurer, S.B., Neri, D., et. al. Purification of Biotinylated
Proteins on Streptavidin Resin: A Protocol for Quantitative Elution.
Proteomics, 4, 2004. pp. 2296-2299.
12. Soller, T., Ringler, M., Wunderlich, M., et. al. Streptavidin Reduces Oxygen
Quenching of Biotinylated Ruthenium(II) and Palladium(II) Complexes.
Journal of Physical Chemistry, 112, 2008. pp. 12824-12826.
13. Zhan, W. and Bard, A.J. Electrogenerated Chemiluminescence. 83.
Immunoassay of Human C-Reactive Protein by Using Ru(bpy3)2+Encapsulated Liposomes as Labels. Analytical Chemistry, 79, 2007. pp. 459463.
14. Zhan, W. and Bard, A.J. Scanning Electrochemical Microscopy. 56. Probing
Outside and Inside Single Giant Liposomes Containing Ru(bpy3)2+. Analytical
Chemistry, 76, 2006. pp. 726-733.
15. Svensson, F.R., Li, M., Nordén, B., et. al. Luminescent DipyridophenazineRuthenium Probes for Liposome Membranes. Journal of Physical Chemistry B,
112, 2008. pp. 10969-10975.
28
CHAPTER 3
Long-Term Stability of Hydrophobicity and Carboxyl Functionality on Polystyrene
Surfaces
3.1: Introduction
3.1.1: Polystyrene: Design of Microfluidic Devices
Polystyrene is a polymer characterized by an alkyl chain with every other carbon
containing a benzyl ring attached to it. Polystyrene replaced glass in the 1960s to study
the interactions between cells and surfaces. It was cheap, easy to manufacture, and cell
cultures would easily adsorb onto the surface when modified1. Modification of
polystyrene surfaces allows for a cheap, disposable way to observe cell interactions2.
Polystyrene has been employed with the used of assays for detection of biological
molecules, even such that detection can occur at low concentrations of the molecule3.
3.1.2: Stability of UV and Ozone Treated Surfaces
The lack of charge in polystyrene makes it a very hydrophobic, nonconducting
surface. Independently, UV and ozonolysis5 have been used to make polystyrene more
hydrophilic. Additionally, other reactions have been used to make polystyrene
surfaces more hydrophilic6,7,8. Research has shown that coupling UV light and ozone
functionalizes the surface with carboxylic acid more efficiently than UV or ozone
independently1. More specifically, this surface coverage is more uniform and
consistent than using UV light and ozone separately1.
29
Following UV and ozone exposure, polystyrene surfaces become functionalized with
carbonyl, alcohol, ether, and ester groups in addition to the carboxylic acid
functionality1. These functional groups can also affect the hydrophobicity of
polystyrene, while not altering the surface charge. It has been shown that following
UV and ozone treatment, polystyrene surface functionality has been maintained for a
long period of time after treatment1. Longer exposure to UV and ozone is effective at
functionalizing the surface, but ultimately unnecessary as carboxylic acid surface
concentration reaches a maximum after ten minutes of exposure1. Additionally, while
the surface chemistry of polystyrene is not altered eight months after exposure, it has
not been determined at what point after treatment did polystyrene reach a saturation
concentration of carboxylic acids. It is also unknown under what storage conditions
may polystyrene is maintained to achieve optimum surface concentration.
In this experiment, polystyrene was UV-treated with ozone gas flow to generate a
more hydrophobic surface and increase the carboxyl surface concentration. The
hydrophobicity and carboxyl surface concentration was characterized by measuring
water contact angles and toluidine blue O (TBO) Assays. Long-term stability of
hydrophobicity and carboxyl surface chemistry was compared for samples stored in
phosphate buffer against samples stored in dry conditions. There was also a
comparison of samples stored at room temperature, refrigeration temperature, and
freezer temperature. Samples were tested for water contact angle and carboxyl surface
concentration by comparing samples that reached equilibrium surface concentrations
stored under different buffers (see chapter 4).
30
3.2: Materials and Methods
3.2.1: Sample Preparation
Polystyrene samples were stored at three different temperatures (23oC, 4oC, and 20oC), in two different storage mediums (dry vacuum sealed bags and 0.1 M
phosphate buffer, pH 7.0), at nine different time points following treatment (1 hour, 5
hours, 24 hours, 48 hours, 4 days, 7 days, 2 weeks, 3 weeks and 4 weeks). Polystyrene
samples were cut from Petri dish plates. Samples were roughly 10 cm2 in area. Eight
pieces were cut for each condition tested. After shaping the samples, the samples were
cleaned via sonication in 50% isopropanol in water at room temperature for ten
minutes. Samples were then rinsed with deionized water and dried with N2. Most of
the dried samples were placed under ultraviolet (UV) light with O3 flown in at
0.5 L/min for ten minutes, followed by a two minute flush with air. Light was applied
using a Jelight UVO-Cleaner, model# 144AX, at a wavelength of 254 nm using a
power of 28 mW/cm2. Light was emitted ~1 cm above the tray surface. Most of these
samples were washed with deionized water and dried with N2.Samples that needed to
be stored were stored immediately after the second drying with N2 gas. Additional
samples tested were polystyrene that was not treated with UV light and ozone,
polystyrene that was treated with both UV light and ozone but that was not washed
after treatment, and polystyrene that was washed after treatment, but not stored in any
medium (immediate testing after last cleaning).
3.2.2: Water Contact Angle Analysis
For each condition, four samples were used for water contact angle analysis. Samples
stored in phosphate buffer were rinsed with deionized water and dried with N2 before
31
contact angles were measured. Five 3 µL droplets were placed on each slide. Droplets
were examined using an LW Scientific Inc. Minivid. Windows Movie Maker was used
to record images of droplets, while the LBASDA plugin to Image-J from the École
Polytechnique Fédéral de Lausanne was used to analyze the contact angles.
Water contact angles were measured using Thomas Young’s equation for surface
wetting:
(2) γl,g cosθ = γl,g – γs,l
Where g, l, and s represent the gas, liquid and solid phases, θ is the water contact
angle, and γ is the excess free energy per unit area at the interface9. This equation was
used to assist in the approximation of the water contact angles with the polystyrene
surface. The program utilizes a spline approach, with set boundary conditions for
control points of the droplet surface and assumes that the reflection of the droplet can
be seen under the glass surface10. The program’s standard error is within three degrees
of the water contact angle.
3.2.3: Toluidine Blue O (TBO) Assay for Quantifying Carboxyl Surface
Concentration
Four samples for each condition (storage temperature, storage medium, and storage
time) were used in the analysis of carboxyl surface functionality. TBO was diluted to a
concentration of 500 µM in pH 10 water for binding to carboxyl functional groups.
Sample surfaces were place in contact with the TBO solution for two hours. After two
hours, each sample was washed three times with pH 10 water to remove any dye that
32
TBO Calibration
4.5
4
y = 3042.8x 0.7834
R2 = 0.9905
Absorbance
3.5
3
2.5
2
1.5
1
0.5
0
0
0.00005
0.0001
0.00015
0.0002
0.00025
Concentration (M)
Figure 3.1 Calibration curve for determination of carboxyl surface concentration on UV treated
polystyrene surfaces. It is assumed that the number of moles in the dye is equal to the number of
moles of carboxylic acid functional groups on the surface of the polystyrene. Surface concentration
calculation was performed by multiplying the dye concentration by the desorbing volume and dividing
over the desorbing area.
did not adsorb onto the surface. Samples were left to air dry for one hour. After
surfaces were dried, areas of approximately 3.4 cm2 were desorbed from each sample
using 50% wt acetic acid. Desorbing area was outlined by polydimethylsiloxane
(PDMS) wells. These wells were cut to contain a base area of about 3.4 cm2. PDMS
sides were cleaned with scotch tape between uses, and stuck to the surface to be
desorbed. Desorption lasted about fifteen minutes. Desorbed samples were measured
for absorbance at a wavelength of 633 nm using a spectrophotometer. A standard
curve was generated using concentrations of 5 µM, 10 µM, 20 µM, 50 µM, 75 µM,
100 µM, 150 µM, and 200 µM (see figure 3.1). Calculation of surface concentration
was performed using the following equation, fitting the curve to a power plot:
33
(3) A = k*cn
A is the absorbance, k and n are constants, and c is the concentration of TBO in the
solution.
3.3: Results and Discussion
3.3.1: Analysis of Controls
Polystyrene surfaces were successfully functionalized as a result of the UV and ozone
treatment. The average water contact angle for a sample that was not treated with UVlight and ozone was 82.0o with a standard deviation of about 6.6o (see figure 3.2).
Samples that were treated with UV-light and ozone, but that had not been washed and
dried after treatment averaged a water contact angle of 11.0o with a standard deviation
of 3.3o. The samples that were treated with UV-light and ozone, and that were washed
after treatment, but not stored in any medium (zero time) averaged a water contact
angle of 42.0o with a standard deviation of 7.7o. These results show that
hydrophobicity significantly decreased as a result of the UV-treatment and ozone, but
that many of the surface functional groups had been modified or washed off by the
wetting of the surface. This is consistent with the results seen in other research11.
Comparison of the non-UV treated samples to the UV-treated samples shows a
dramatic increase in the functionalization of carboxyl groups on the polystyrene
surface (see figure 3.3) Average carboxyl surface concentration for non-UV treated
polystyrene was approximately 0.3 nmol/cm2 with a standard deviation of 46
pmol/cm2. This is significantly less than the UV and ozone treated samples that were
not rinsed and the UV and ozone treated samples that were rinsed and not stored.
34
Water Contact Angle (Degrees)
Water Contact Angles for Polystyrene Samples
100
90
80
70
60
50
40
30
20
10
0
No UV
No Wash
Not Stored
Treatment
Figure 3.2 Average water contact angles for non-stored polystyrene. Samples tested included
polystyrene not treated with UV and ozone, polystyrene that had been treated for ten minutes, but had not
been rinsed, and samples that had been rinsed after washing and were immediately tested for
hydrophobicity.
Carboxyl surface concentration for treated and unwashed samples averaged 15.8
nmol/cm2 with a standard deviation of 0.23 nmol/cm2. Carboxyl surface concentration
for treated, washed, but not stored samples averaged 12.4 nmol/cm2 with a standard
deviation of 1.24 nmol/cm2.
3.3.2: Forty Eight Hours After Initial Treatment
Average water contact angles were calculated for each storage condition measured
(time after treatment, storage temperature, and storage medium; see figure 3.4, and
table 3.1). Overall, hydrophobic rebound occurred most rapidly in the first hour after
treatment, leveling off after 24 hours of treatment. Water contact angles spiked from
42o to around 55o after the first day, with a slight decrease towards the second day.
Treatment temperature appeared not affect the water contact angle significantly during
the short time after UV-treatment with ozone. For both the phosphate stored samples
35
Surface Concentration (nmol/cm^2)
Carboxyl Surface Chemistry
16
14
12
10
8
6
4
2
0
No UV
No Wash
Not Stored
Surface Treatment
Figure 3.3 Average carboxylic acid surface concentration for non-stored polystyrene. Samples
tested included polystyrene not treated with UV and ozone, polystyrene that had been treated for ten
minutes, but had not been rinsed, and samples that had been rinsed after washing and were immediately
tested for hydrophobicity.
and the dry stored samples, hydrophobic rebound within the hour after treatment was
slowest for the 23oC samples, but by the fifth hour, the variation amongst the
temperatures had settled. Comparison of the samples stored under dry conditions to
the samples stored in phosphate buffer shows that there is not a significant difference
in hydrophobic rebound between both storage media at any of the observed
temperatures.
Water Contact Angle at Various Temperatures
Water Contact Angle (Degrees)
Water Contact Angle (Degrees)
Water Contact Angles at Various Storage Temperatures
70
60
50
23
40
4
30
-20
20
10
0
70
60
50
23
40
4
30
-20
20
10
0
0
1
5
24
48
0
Time After UV-Treatment (Hours)
1
5
24
48
Time After UV-Treatment (Hours)
Figure 3.4 Average water contact angle for polystyrene 1 hr, 5 hr, 24 hr, and 48 hr after UV and
ozone treatment. Blue bars represent samples stored at 23oC, red bars represent samples stored at 4oC
and green bars represent samples stored at -20oC. The graph on the left depicts samples stored in dry
conditions while the graph on the right represents samples stored in 0.1 M pH 7 sodium phosphate buffer.
36
Carboxyl Surface Chemistry
Surface Concentration (nmol/cm^2)
Surface Concentration (nmol/cm^2)
Carboxyl Surface Chemistry
18
16
14
12
23
10
4
8
-20
6
4
2
16
14
12
10
23
8
4
6
-20
4
2
0
0
0
1
5
24
0
48
1
5
24
48
Time after Treatment (Hours)
Time After Treatment (Hours)
Figure 3.5 Average carboxyl surface concentration for polystyrene 1 hr, 5 hr, 24 hr, and 48 hr after
UV and ozone treatment. Blue bars represent samples stored at 23oC, red bars represent samples stored
at 4oC and green bars represent samples stored at -20oC. The graph on the left depicts samples stored in
dry conditions while the graph on the right represents samples stored in 0.1 M pH 7 sodium phosphate
buffer.
Carboxyl surface concentration appears to decrease rapidly within the first hour for
polystyrene stored in phosphate buffer (see figure 3.5 and table 3.2). This dramatic
reduction was seen for all of the temperatures studied. In contrast, carboxyl surface
concentration for dry stored samples was greater for the 5 hr time point than it was
right after UV and ozone treatment. This disparity seen between the one hour and five
hour can be attributed to the leakiness of the PDMS wells used to confine the
desorbing area. Regardless, dry stored samples consistently maintained a greater
carboxyl surface concentration than the polystyrene stored in phosphate buffer.
Additionally, the -20oC samples stored in phosphate buffer had less of a reduction in
carboxyl surface chemistry than the samples stored in the same medium but at warmer
temperatures. This was seen at all time points within the first two days except one
hour after treatment. However, the variation in the carboxyl surface concentration is
relatively high, so the difference is ultimately not that great between the samples
stored at different temperatures.
3.3.3: Seven Days After Initial Treatment
Water contact angle over the first week appeared to have reached a maximum around
day 4 for the dry stored samples, while the maximum angle was reached after 24 hours
37
Water Contact Angle at Various Storage Temperatures
Water Contact Angle (Degrees)
Water Contact Angle (Degrees)
Water Contact Angles at Various Storage Temperatures
70
60
50
23
40
4
30
-20
20
10
0
70
60
50
23
40
4
30
-20
20
10
0
0
1
2
3
4
5
6
7
8
0
Time After UV-Treatment (Days)
1
2
3
4
5
6
7
8
Time After UV-Treatment (Days)
Figure 3.6 Average water contact angle for polystyrene 24 hr, 48 hr, 4 days, and 7 days after UV
and ozone treatment. Blue lines represent samples stored at 23oC, red lines represent samples stored at
4oC and green lines represent samples stored at -20oC. The graph on the left depicts samples stored in dry
conditions while the graph on the right represents samples stored in 0.1 M pH 7 sodium phosphate buffer.
of storage for the phosphate stored samples (see figure 3.6 and table 3.1). Most of the
water contact angles measured averaged between 50 and 55o, with none falling outside
the range of 47-60o. This indicates an overall hydrophobic rebound from the original
measurement, but with leveling off occurring significantly less than that of
polystyrene that was not exposed to UV-light and ozone simultaneously. Comparing
temperatures shows that contact angles were greatest for the 23oC stored samples, with
a slightly less contact angle for the 4oC and -20oC stored samples, though comparison
of the two cooler temperatures shows little difference in the contact angle. The
exception to this occurred four days after treatment for the dry stored samples and 24
hours after treatment for the phosphate stored samples. Comparison of the dry stored
samples to the phosphate stored samples during the first week after treatment showed
little variation between samples. Additionally, water contact angle appeared to be
decreasing by end of the first week, approaching a value of around 50o.
Looking at samples after one week of treatment shows a leveling off of carboxyl
surface concentration for polystyrene stored at all temperatures and in both mediums
(see figure 3.7 and table 3.2). Dry stored samples appeared to have leveled off at 17
nmol/cm2, while -20oC samples stored in pH 7.0 phosphate buffer maintained a
surface concentration of approximately 2 nmol/cm2. Additionally, surface
38
Carboxyl Surface Chemistry
Surface Concentration (nmol/cm^2)
Surface Concentration (nmol/cm^2)
Carboxyl Surface Chemistry
20
18
16
14
12
23
10
4
8
-20
6
4
2
0
16
14
12
10
23
8
4
6
-20
4
2
0
0
1
2
3
4
5
6
7
8
0
Time After Treatment (Days)
1
2
3
4
5
6
7
8
Time After Treatment (Days)
Figure 3.7 Average carboxyl surface concentration for polystyrene 24 hr, 48 hr, 4 days, and 7
days after UV and ozone treatment. Blue lines represent samples stored at 23oC, red lines represent
samples stored at 4oC and green lines represent samples stored at -20oC. The graph on the left depicts
samples stored in dry conditions while the graph on the right represents samples stored in 0.1 M pH 7
sodium phosphate buffer.
concentrations of samples stored at 4oC and 23oC at room temperature averaged a
surface concentration of less than 1 nmol/cm2. This is consistent with the results of the
first 48 hours, showing that samples stored in dry, vacuum-sealed bags maintain a
higher carboxyl surface concentration than samples stored in phosphate buffer.
Additionally, the higher concentration with the lower temperature in the phosphate
samples is more pronounced during this duration after treatment. The variation
between surface concentrations is less with these samples than it was with the one
hour and 5 hour samples. Thus, -20oC storage seems to have more favorable shortterm maintenance of carboxyl surface functionality than either the refrigerated or
room-temperature stored samples.
3.3.4: Four Weeks After Initial Treatment
Overall, there was an increase in the measured water-contact angle over the four week
period after UV and ozone treatment (see figure 3.8 and table 3.1). However,
hydrophobic rebound mostly leveled off after the second week following
measurements. Comparison of the samples of similar temperatures shows greater
variation amongst the dry stored samples. Specifically, the samples stored at higher
39
Water Contact Angle At Various Storage Temperatures
70
Water Contact Angle (Degrees)
Water Contact Angle (Degrees)
Water Contact Angle at Various Storage Temperatures
60
50
23
40
4
30
-20
20
10
0
70
60
50
23
40
4
30
-20
20
10
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0
0.5
1
Time After UV-Treatment (Weeks)
1.5
2
2.5
3
3.5
4
4.5
Time After UV-Treatment (Weeks)
Figure 3.8 Average water contact angle for polystyrene 7 days, 14 days, 3 weeks, and 4 weeks after
UV and ozone treatment. Blue lines represent samples stored at 23oC, red lines represent samples stored
at 4oC and green lines represent samples stored at -20oC. The graph on the left depicts samples stored in
dry conditions while the graph on the right represents samples stored in 0.1 M pH 7 sodium phosphate
buffer.
temperatures appeared to have greater hydrophobic rebound. However, all the
measurements still showed that the average contact angle was significantly less than
that of the non-UV treated sample; none of the average water contact angles measured
greater than 60o. For the phosphate stored samples, variation was less in the water
contact angles measured. The values measured were similar to that of the water
contact angles measured for the 4oC and the -20oC samples stored under dry
conditions. Overall, hydrophobic rebound remains stable weeks after UV and ozone
treatment for both storage media and at all the temperatures (though lower
temperatures appear to be more preferable than higher temperatures for maintaining
lower hydrophobicity).
Carboxyl Surface Chemistry
Surface Concentration (nmol/cm^2)
Surface Concentration (nmol/cm^2)
Carboxyl Surface Chemistry
20
18
16
14
12
23
10
4
8
-20
6
4
2
0
16
14
12
10
23
8
4
6
-20
4
2
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0
Time After Treatment (weeks)
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Time After Treatment (weeks)
Figure 3.9 Average carboxyl surface concentration for polystyrene 7 days, 14 days, 3 weeks, and 4
weeks after UV and ozone treatment. Blue lines represent samples stored at 23oC, red lines represent
samples stored at 4oC and green lines represent samples stored at -20oC. The graph on the left depicts
samples stored in dry conditions while the graph on the right represents samples stored in 0.1 M pH 7
sodium phosphate buffer.
40
Looking up to four weeks of storage, it can be seen that a greater surface concentration
is maintained in the dry stored samples than in the phosphate stored samples (see
figure 3.9 and table 3.2). Samples at the two and three week time points appear to have
a lower carboxyl surface concentration than those one week and four weeks after
treatment. This variation is likely due to the leakiness of the PDMS wells. The
opposite was seen with the phosphate stored samples, where the concentration
increased into the second week samples, but leveled off after the third week. The 20oC polystyrene samples stored in phosphate buffer had greater carboxyl surface
concentration than the other samples stored in the phosphate buffer until the fourth
week, which saw a huge drop in the surface concentration of the frozen samples. Until
this time point, -20oC stored samples had a higher carboxyl surface concentration than
the room temperature stored and refrigerated polystyrene samples.
Conclusions and Future Work
The results of the TBO Assay and water contact angle analysis show that carboxyl
surface functionality can be well maintained weeks after treatment with UV-light and
ozone. It also shows that surface saturation for both carboxylic acid functionality and
water contact angle can occur within as little as one hour after treatment. Additionally,
temperature of storage seems to have little effect on the water contact angle of samples
stored in both mediums. This shows that if a surface conjugation needed to occur
between 23oC and -20oC, it could without the reduced functionality of the device.
The results also show that samples stored in a dry medium provide better storage for
UV and ozone treated polystyrene than samples stored in 0.1 M pH 7 sodium
phosphate buffer. While water contact angle is maintained, carboxylic acid surface
41
concentration decreases. Further more, samples stored in phosphate buffer are also
more temperature sensitive than samples stored in water, as samples stored at -20oC
maintained some carboxyl surface functionality, while virtually none of the room
temperature or refrigerated samples had carboxyl groups on them past the first day.
Dry stored samples showed no consistency as to when the surface concentration or the
water contact angle was greatest for a given temperature.
Future experiments involve other modifications that may be done with storage. When
observing samples stored in sodium phosphate buffer, there may be a specific timing
as to when saturation is reached, and how rapidly the surface concentration drops off.
Additionally, dry stored samples can be tested to see if moisture sensitivity can affect
the hydrophobicity and carboxyl surface concentration of UV treated polystyrene.
42
Table 3.1 Average water contact angles for UV and ozone treated polystyrene samples stored over
a four week duration.
Average
Standard
Dry Stored Deviation
Average
Phosphate
Buffer
Stored
Standard
Deviation
Average
Standard
Dry Stored Deviation
Average
Phosphate
Buffer
Stored
Standard
Deviation
Temp 1 hr
5 hrs 24 hrs 48 hrs 4 days
49.9
52.4
53.0
55.4
23
57.4
54.7
53.2
47.3
50.8
4
59.2
52.1
51.5
48.3
50.3
-20
54.8
6.0
4.9
5.3
3.5
23
3.8
3.8
4.1
6.2
4.1
4
5.2
2.4
5.1
7.2
6.0
-20
3.4
50.7
51.3
57.3
55.9
23
54.4
52.7
52.1
58.9
52.0
4
52.8
51.7
54.5
57.1
52.5
-20
50.8
2.2
4.2
3.8
4.6
23
3.3
2.1
3.7
2.9
4.6
4
4.4
2.7
4.9
4.5
2.8
-20
4.0
Temp 7 days 2 wks 3 wks 4 wks
54.2
58.5
56.8
58.9
23
49.5
57.1
54.9
55.0
4
50.8
51.5
52.6
52.5
-20
4.9
5.1
5.2
4.4
23
4.6
3.4
6.9
2.9
4
3.0
4.0
3.7
3.0
-20
51.5
55.3
54.9
56.5
23
50.5
55.2
56.9
59.5
4
50.1
53.8
57.7
59.0
-20
7.2
4.0
5.7
3.6
23
3.4
4.4
4.7
3.5
4
3.6
3.1
4.6
3.6
-20
43
Table 3.2 Average carboxyl surface concentration for UV and ozone treated polystyrene samples
stored over a four week duration.
Average
Dry
Stored
Standard
Deviation
Average
Phosphate
Buffer
Stored
Standard
Deviation
Average
Dry
Stored
Standard
Deviation
Average
Phosphate
Buffer
Stored
Standard
Deviation
Temp 1 hr
5 hr
24 hr
48 hr
4 days
5.9
15.8
9.2
10.7
17.4
23
7.1
15.6
8.9
11.1
17.1
4
4.0
15.6
7.5
9.2
16.3
-20
2.22
0.20
2.24
1.45
0.11
23
1.99
0.40
0.35
1.84
0.38
4
1.34
0.54
1.32
2.23
0.75
-20
2.6
5.6
2.0
1.6
0.3
23
1.7
5.8
2.1
2.1
0.2
4
2.1
8.2
2.2
2.2
1.7
-20
0.87
1.50
0.20
0.13
0.05
23
0.57
0.82
0.07
0.26
0.01
4
0.13
1.10
0.73
0.30
0.51
-20
Temp 7 days 2 wks
3 wks
4 wks
16.9
13.5
13.5
17.2
23
17.0
11.8
12.3
17.7
4
17.0
13.3
13.0
17.4
-20
0.34
1.42
0.42
0.38
23
0.15
0.99
1.24
0.31
4
0.09
0.49
0.96
0.27
-20
0.2
5.2
4.1
4.8
23
0.2
5.7
5.5
5.2
4
1.7
7.1
6.1
0.4
-20
0.03
0.93
0.66
0.72
23
0.02
0.56
0.28
0.38
4
0.35
0.62
0.02
0.19
-20
44
REFERENCES
1. Callen, B.W., Ridge, M.L. Lahooti, S. Remote Plasma and Ultraviolet-Ozone
Modification of Polystyrene. J. Vac. Sci. Tehnol. A., 13, 4, 1995. pp. 20232029.
2. Curtis, A.S.G., Forrester, J.V., McInnes, C., et. al. Adhesion of Cells to
Polystyrene Surfaces. J. Cell Biol., 97, 1983. 1500-1506.
3. Do, J., Ahn, C.H. A Polymer Lab-on-a-Chip for Magnetic Immunoassay with
On-Chip Sampling and Detection Capabilities. Lab Chip, 8, 2008. pp. 542549.
4. Bliznyuk, V., Assender, H., Briggs, A., et. al. Surface Glass Transition
Temperature of Amorphous Polystyrene Measured by SFM. APS Meeting,
March 2001.
5. Jellinek, H.H.G. and Kryman, F.J. Gas Analysis by Polymer Chain Scission:
Ozonolysis of Polystyrene. Environmental Science and Technology, 1, 1967.
pp. 658-660
6. Callen, B.W., Sodhi, R.N.S., Shelton, R.M., et. al. Behavior of Bone Cells
Characterized on Polystyrene Surfaces. Journal of Biomedical Materials
Research, 27, 1993. pp. 851-859
7. Curtis, A.S.G., Forrester, J.V., and Clark, P. Substrate Hydroxylation and Cell
Adhesion. Journal of Cell Science, 86, 1986. pp. 9-25.
8. Ertel, S.I., Chilkoti, A., Horbett, T.A., et. al. Endothelial Cell Growth on
Oxygen-Containing Films Deposited by Radio-Frequency Plasmas: the Role of
Surface Carbonyl Groups. Journal of Biomaterials Science: Polymer Edition,
3, 1991. pp. 163-183.
9. T. Young. An Essay on the Cohesion of Fluids. Philos. Trans. R. Soc. Lond.,
1905. pp. 65-87.
45
10. Stalder, A.F. Kulik, G., Sage, D. A Snake-Based Approach to Accurate
Determination of Both Contact Points and Contact Angles. Colloids and
Surfaces A: Physicochem. Eng. Aspects, 286, 2006. pp. 92-103.
11. Zhang, D., Dougal, S.M., and Yeganeh, M.S. Effects of UV Irradiation and
Plasma Treatment on a Polystyrene Surface Studied by IR-Visible Sum
Frequency Generation Spectroscopy. Langmuir, 16, 2000. pp. 4528-4532.
46
CHAPTER 4
The Affects of Heating, Surface Coating, and Buffer Storage on Hydrophobicity and
Carboxyl Functionality of UV-Treated Polystyrene and PMMA Surfaces
4.1: Introduction
4.1.1: PMMA: Design of a Microfluidic Device
Poly(methyl methacrylate) (PMMA) is an alkyl polymer characterized by which every
other carbon forming methyl acetate group on the surface. These repeating ester
groups within PMMA allow for it to have some reactivity, while maintaining a mostly
stable structure. It has been shown that PMMA conjugated with graphite can be more
electrically conductive, and allow for efficient transport of solutes formed in channels
of microfluidic devices made of PMMA1. Additionally, PMMA is used because of its
transparency and low autofluorescence2.
Like polystyrene, PMMA is a very hydrophobic material before UV-treatment. After
UV treatment with ozone, the PMMA surface polymer becomes replaced with alcohol,
ether, ester, carbonyl, and carboxyl groups. PMMA be used effectively in the
development of microfluidic devices because of its good surface bonding at low
temperatures3. Additionally, PMMA is cheap and easy to make, which has allowed it
to replace glass in the formation of many devices. Due to the good conjugation
chemistry between carboxylic acids and many biomolecules, assays can be coupled
with the microfluidics, forming devices that can serve multiple functions4.
47
4.1.2: Thermal and Chemical Effects on Carboxyl Surface Chemistry and
Hydrophobicity
In the formation of microfluidic devices, polystyrene or PMMA may be heated to
facilitate the creation of the channels in the device, or fort bonding purposes5. This can
be done as an alternative to formation of channels using polydimethylsiloxane
(PDMS), which is better suited for the formation of channels requiring hydrophobic
small molecule attachment or conjugation6. While polystyrene has a melting
temperature of approximately 240oC5, PMMA has a melting temperature of ~130135oC7. While heating for channel formation usually occurs at 80-100oC, polystyrene
glass can warp or dissolve around 95oC5.8. Conversely, the glass melting temperature
(or glass transition temperature) of PMMA is approximately equal to its melting
temperature9. Additionally, any heat applied to polystyrene in the presence of water
can distort the shape of the device, and ultimately impair the ability of the channels to
form properly. However, altering the glass transition temperature has been shown to
allow for bonding between surfaces without altering the geometry of the channel10.
Glass melting temperatures may also vary depending on the thickness of the device
and the presence of other materials within the compound (other plastics, impurities,
etc)11.
Surface wetting can often be used to facilitate the bonding of two surfaces of a
device12. After polystyrene is treated with UV light and ozone, the surface will be
bound to another surface, forming the device. This would entail the surface being
coated with acetone or acetyl acetone, followed by immediate binding between the
treated surface and the other surface to be bound13. Additionally, acetyl acetone can
48
form cyclic compounds with many metals, which can promote the formation of
electrodes onto a PMMA or polystyrene surface14.
While the development of microfluidic devices using polystyrene is well known, it is
unknown if any of these steps effect the hydrophobicity and carboxyl surface
functionality of PMMA or polystyrene. It is important to know if these factors affect
surface chemistry as it could impact the methods of developing devices with
carboxylic acids functionalized on the surface. Additionally, while surface chemistry
has been shown to affect glass transition temperature15, it is unknown whether or not
glass transition temperature can affect surface chemistry, specifically, if it affects
carboxylic acid surface chemistry.
In this experiment, several factors affecting carboxyl surface concentration and
hydrophobicity were compared. First, UV and ozone treated PMMA and polystyrene
were heated to three different temperatures and tested to see if the heating reduced the
carboxyl surface concentration. Next, polystyrene was coated with a surface bonding
solvent (acetyl acetone) to determine if that also reduced carboxyl surface
concentration. Last, as an extension of the research described in chapter 3, samples
were stored in different buffers and at different pH for a short amount of time to
determine if carboxyl surface concentration was reduced, and if hydrophobicity
increased. These results would ultimately show the optimum storage conditions for
UV-treated polystyrene, the best conjugation buffer for any biomolecule to be
immobilized to the polystyrene surface, and how factors used for surface bonding
would affect the surface functionality and hydrophobicity.
49
4.2: Materials and Methods
4.2.1: Sample Preparation
Eight samples were prepared for each condition observed. Conditions tested included
a comparison of PMMA to Polystyrene, and three temperatures (90oC, 95oC, and
100oC) used in the formation of channels in microfluidic devices. PMMA samples
were cut to 2 cm by 3 cm samples. Polystyrene samples were cut as described in
chapter 4. Three control sample types were also prepared: non-UV treated samples,
samples that had been treated with UV and ozone, but had not been washed, and
samples that had been washed but without heating occurring. Samples were cleaned
via ten minute sonication in 50% isopropanol in water at room temperature. Samples
were then rinsed with deionized water and dried with N2 gas. Samples were exposed to
UV light and ozone for ten minutes with an ozone flow rate of 0.5 L/min. This was
followed by a two minute flush. Samples to be used for temperature experiments were
heated to the desired temperature on a (insert name of machine here) for five minutes.
Following the heating, samples were washed with deionized water and dried with N2
gas to be used for experiments.
Spin coated samples were treated with UV and ozone as described in chapter 4. After
the second wash and dry with deionized H2O and N2, polystyrene samples (UV treated
and non-UV treated were coated) with acetyl acetone using a CHEMAT Technology
Spin coater KW-4A. Droplets were placed on the polystyrene surface and were spun at
1000 rpm for 4.5 seconds.
Samples stored in solution buffers were placed in five different buffers and at basic,
neutral, or acidic pH for each buffer. Samples stored in water or in 0.1 M PBS were
50
tested for water contact angle and hydrophobicity at pH 5, 7, and 9. Samples stored in
0.1 M HEPES, 0.1 M Sodium Borate, or 1 M Tris were stored at pH 6, 7, and 8.
Buffer and pH samples were stored for 1 hour at 23oC following treatment before
being tested for water contact angle and surface carboxylic acid concentration.
4.2.2: Water Contact Angle Analysis
Water Contact Angles were analyzed as shown in Chapter 3: Long-Term Stability of
Hydrophobicity and Carboxyl Functionality on Polystyrene Surfaces. Four samples
with five droplets per sample were used for each condition (channel material and
temperature of heating).
4.2.3: TBO Assay for Quantified Carboxyl Surface Concentration
TBO Analysis was performed as explained in Chapter 3: Long-Term Stability of
Hydrophobicity and Carboxyl Functionality on Polystyrene Surfaces. The only
modification made to the analysis was for the PMMA surfaces, the desorbing area
covered the entire 6 cm2 sample. Consequentially, surface concentration had to change
based on that model.
4.3: Results and Discussion
4.3.1: PMMA and Polystyrene: Thermal Bonding
Like polystyrene, PMMA shows a similar trend in comparing non-UV treated ozone to
UV-treated ozone (see figure 4.1) However, the extremity of the values was much
more significant for the polystyrene samples than the PMMA samples. PMMA that
51
was not treated with UV averaged a water contact angle of 66.0o with a standard
deviation of 3.6o. PMMA that was treated with UV and ozone but that had not been
rinsed following treatment average a water contact angle of 28.4o with a standard
deviation of 3.4o. While these PMMA samples showed less variation in contact angles,
the reduction in hydrophobicity following UV treatment was significantly less than
that seen in polystyrene. Furthermore, samples that had been rinsed with water after
treatment without heating experienced an average water contact angle of 36.2o with a
standard deviation of 4.1o. This value is significantly less that of rinsed, UV-treated
polystyrene. Additionally, the rebound that occurs due to washing is much less than
that of polystyrene. Washing shows an average increase in water contact angle of 7.8o
for PMMA and 31.0o for polystyrene. These results indicate that UV-treatment with
ozone may affect the surface bonding chemistry of polystyrene more than in PMMA.
Comparison of heated samples to unheated samples shows a complete hydrophobic in
Water Contact Angle (Degrees)
Water Contact Angles for PMMA Samples
90
80
70
60
50
40
30
20
10
0
No UV
No Wash
Not Heated
Treatment
Figure 4.1 Water contact angle analysis for PMMA control samples. Included in the control samples
are untreated samples, PMMA samples treated with UV light and ozone, but not washed, and PMMA
samples that were washed with deionized water following treatment, but were not heated before washing.
52
Water Contact Angle (Degrees)
Water Contact Angles for Samples Heated for Five Minutes
80
70
60
50
Polystyrene
40
PMMA
30
20
10
0
No Heat
90
95
100
Temperature of Heat (degrees Celsius)
Figure 4.2 Water contact angle analysis for UV and Ozone treated PMMA and polystyrene heated
samples. Blue bars represent polystyrene, while red bars represent PMMA. All samples were UV and
ozone treated, some of the samples were heated to 90, 95, or 100 oC for five minutes before a final rinse
with deionized water.
heating with PMMA (see figure 4.2 and table 4.1). For all cases of heating, average
water contact angle was within range of the water contact angle of non-treated
samples. The lowest contact angles measured occurred at 90oC heating, while the
highest contact angles measured came from the 95oC heated samples, with the 100oC
heated samples having similar contact angles to that of non-UV treated samples. In
comparison, polystyrene samples experienced almost negligible hydrophobic rebound
upon being heated (see figure 4.2 and table 4.1). Though all the samples did show
some hydrophobic rebound after heating, the average value for each heated sample
was less than that of one hour storage time for all different temperature and storage
conditions. Additionally, results show that there is little effect that the heating
temperature has on the water contact angle. This was true for both polymers, but even
more so for polystyrene where the difference between the greatest and least of these
contact angles was about 1.5o.
53
Surface Concentration (nmol/cm^2)
Carboxyl Surface Chemistry, PMMA Samples
12
10
8
6
4
2
0
No UV
No Wash
Not Stored
Surface Treatment
Figure 4.3 Carboxylic acid surface quantification for PMMA control samples. Included in the
control samples are untreated samples, PMMA samples treated with UV light and ozone, but not
washed, and PMMA samples that were washed with deionized water following treatment, but were not
heated before washing.
Like polystyrene, carboxyl surface concentration increased on PMMA surfaces after
applying UV light and ozone for ten minutes (see figure 4.3). However, hydrophobic
rebound did not occur when the PMMA samples were washed. Comparison shows that
UV-treated PMMA (washed and unwashed) had a carboxyl surface concentration of
9.8 nmol/cm2, while untreated samples had 0.098 nmol/cm2 carboxyl surface
concentration. Ultimately, the UV-treatment with ozone did functionalize the surface
with carboxylic acids and washing did not affect carboxylic acid functionality.
Heating of polystyrene surfaces seemed to have decreased carboxyl surface
concentration, while heating of PMMA surfaces did not effect carboxyl surface
concentration (see figure 4.4 and table 4.2). After heating, carboxylic acid surface
concentration on polystyrene appears to drop from approximately 12 nmol/cm2 to 7
nmol/cm2. However, measured surface concentrations of all PMMA surfaces
following heating fell between 9.7 and 10 nmol/cm2. Thus, both surfaces will still
54
Surface Concentration (nmol/cm^2)
Carboxyl Surface Chemistry, Heated Samples
16
14
12
10
Polystyrene
8
PMMA
6
4
2
0
No Heat
90
95
100
Temperature of Heating (Celsius)
Figure 4.4 Carboxylic acid surface quantification for UV and Ozone treated PMMA and
polystyrene heated samples. Blue bars represent polystyrene, while red bars represent PMMA. All
samples were UV and ozone treated, some of the samples were heated to 90, 95, or 100 oC for five
minutes before a final rinse with deionized water.
retain most of the carboxyl surface functionality, with UV-treated PMMA being
completely unaffected by heat.
4.3.2: Polystyrene and Solvent Bonding
Comparison of spin coated and non-spin coated samples showed complete
hydrophobic rebound following spin-coating (see figure 4.5). UV treated and spin
coated samples had an average water contact angle of 84.9o, while non-UV-treated
samples had an average water contact angle of 81.7o. Ultimately, there was no
significant difference between non-UV-treated and acetyl acetone coated samples,
UV-treated and acetyl acetone coated samples, and non-UV-treated samples.
Samples spin coated with acetone showed a reduction in carboxyl surface
functionality compared to UV-treated and washed samples (see figure 4.6). Non-UV
treated acetyl acetone covered samples averaged a surface concentration of 0.22
55
Water Contact Angle (Degrees)
Water Contact Angle for Acetyl Acetone Coated Samples
100
90
80
70
60
50
40
30
20
10
0
Non UV
UV
Treatment
Figure 4.5 Water contact angle analysis for polystyrene with a surface coating of acetyl acetone.
Comparison of non treated against UV and ozone treated samples.
nmol/cm2 while UV-treated, acetyl acetone coated samples averaged a surface
concentration of 6.5 nmol/cm2. While the error range on the untreated samples was
low, UV-treated samples had a standard deviation of 4.4 nmol/cm2. Thus, there is still
a greater carboxyl surface concentration for UV-treated samples. However, there is a
significant reduction in the surface concentration after coating the surface with acetyl
acetone as the functional groups will be covered by the solvent.
The spin coated samples did not appear uniformly blue. This suggests that acetyl
acetone did not completely coat the surface of the sample. This non-uniform coating
may be responsible for the large variance observed by the UV-treated and acetyl
acetone coated samples
4.3.3:Buffer and pH Experiments
56
Surface Concentration (nmol/crm^2)
Carboxyl Surface Chemistry, Acetyl Acetone Spindown
samples
12
10
8
6
4
2
0
No UV
UV
Treatment
Figure 4.6 Carboxylic acid surface quantification for polystyrene with a surface coating of acetyl
acetone. Comparison of non treated against UV and ozone treated samples
For pH tested samples, H2O and tris buffer stored samples maintained the lowest water
contact angles, all of which averaged between fifty three and fifty six degrees (see
figure 4.7 and table 4.3). Additionally, for all samples except for water and sodium
borate, water contact angle increased with pH. For sodium borate, water contact angle
was greatest with the pH 7 samples. For the H2O stored samples, water contact angle
did not vary much with pH, though pH 9 H2O had the lowest average water contact
angle measured amongst the water stored samples. pH 9 H2O and pH 6 and 7 tris
appeared to have the least hydrophobic rebound of any of the samples. Sodium borate
and moderate to high pH HEPES and PBS were the buffers that caused the most
hydrophobic rebound. Overall, the HEPES, sodium borate, and PBS buffers caused
significantly more hydrophobic rebound than sodium phosphate stored, H2O stored,
tris stored, or dry stored samples, one hour after treatment. It appears that the other
buffers and storage mediums did not significantly alter water contact angle.
Samples stored in various buffers indicated that acidic and neutral H2O and acidic
PBS can be used for maintaining carboxyl surface chemistry best (see figure 4.8 and
57
Water Contact Angle (Degrees)
Water Contact Angle, Various Buffers and pH
80
70
60
Water
50
PBS
40
HEPES
30
Sodium Borate
Tris
20
10
0
5
6
7
8
9
pH
Figure 4.7 Water contact angle analysis for UV and ozone treated polystyrene stored in various
solutions for 1 hour at either acidic, neutral or basic pH. Water and PBS buffer stored samples were
analyzed at pH 5, 7, and 9. HEPES buffer, sodium borate buffer, and tris buffer stored samples were
analyzed at pH 6, 7, and 8.
table 4.4). HEPES and sodium borate stored samples appeared to have cut the surface
concentration in half. Samples stored in tris buffer at a pH of 7 maintained a carboxyl
surface concentration significantly higher than that of samples stored in HEPES and
sodium borate and that of acidic or basic tris. Basic water and neutral and acidic PBS
have carboxyl surface functionality similar to that of UV-treated polystyrene stored in
pH 7 tris buffer. Overall, samples stored in acid or neutral water, acidic PBS, and in
dry vacuum sealed bags retain the most carboxylic acid functionality for samples to be
used shortly after treatment.
4.4: Conclusions and Future Work
The heating of PMMA and polystyrene for surface bonding and channel formation did
affect the surface chemistry. While there appeared to be no correlation between
heating temperature and water contact angle, heating of the PMMA surface did
58
18
16
14
(nmol/cm^2)
Carboxyl Surface Concentration
Carboxyl Surface Chemistry, Varied Buffers and pH
Water
12
PBS
10
HEPES
8
Sodium Borate
6
Tris
4
2
0
5
6
7
8
9
pH
Figure 4.8 Carboxylic acid surface quantification for UV and ozone treated polystyrene stored in
various solutions for 1 hour at either acidic, neutral or basic pH. Water and PBS buffer stored
samples were analyzed at pH 5, 7, and 9. HEPES buffer, sodium borate buffer, and tris buffer stored
samples were analyzed at pH 6, 7, and 8.
increase hydrophobicity. However, with carboxyl surface chemistry remaining
constant for all three thermal bonding temperatures, a likely event of the heating is that
the surface chemistry was altered, but that carboxyl surface chemistry remained
unaffected. It is likely that other hydrophilic groups were removed from the surface,
such as alcohol and ether groups. It has been shown that in polystyrene, UV and ozone
treatment can form functional groups other than carboxylic acids16.
On the other end of the spectrum, polystyrene did not become more hydrophobic, but
it did lose its surface functionality. However, the loss of functionality was also not
correlated to temperature. Additionally, the final surface concentration after heating
was still greater than 50% of the original surface concentration. Though there was a
dramatic reduction in the carboxyl surface concentration, the reduction should not be
enough to significantly alter the ability for proteins to be adsorbed onto the surface.
This is further shown through the retention in hydrophobicity.
59
Acetyl acetone is a very hydrophobic solvent. As such, it is not surprising to see that
there was a great reduction in the carboxyl surface concentration, as well as an
increase in water contact angle to the point of having the same hydrophobicity as
untreated polystyrene. The results of the TBO assay show a large span of possible
surface concentrations, indicating that nothing can be concluded about the effect of
acetyl acetone coating on carboxyl surface chemistry. If there is a reduction in the
surface chemistry, this may not affect surface bonding. Since surface bonding occurs
immediately after coating, channels would be washed out before acetyl acetone firmly
dries onto the channel surface. If this occurs, surface chemistry would not be altered
within the channels if acetyl acetone is properly flushed out. Determination of spin
coating with acetyl acetone immediately followed by washing with water should
determine whether or not the solvent bonder would affect the channel’s surface
chemistry.
The presence of buffers indicates the reactivity of the carboxylic acid surface.
Carboxyl surface functionality was well maintained only in samples stored in dry
conditions, pH 5-7 water, and pH 5 PBS buffer. Additionally, pH 7-9 PBS buffer, pH
9 H2O, and pH 7 tris buffer maintained more than 50% functionality. Looking at
hydrophobicity, samples stored in dry conditions, water, 0.1 M phosphate buffer, and
tris buffer increased hydrophobicity the least. These results indicate that the best suited
storage for UV and ozone treated polystyrene is under dry conditions or in pH 5-7
water. The hydrophobic rebound and loss of functionality observed in the sodium
borate and HEPES buffers show that they are inadequate for polystyrene storage.
Additionally, low pH seems to be favorable for retaining hydrophobicity. This makes
sense, as the presence of free floating hydrogen ions would increase the surface charge
60
of the negatively charged carboxyl groups. PBS pH 5 is the buffer most suitable for
chemical modification of polystyrene. More buffers should be tested for surface
functionality and hydrophobicity, and the pH trend in water contact angle should also
be investigated with other buffers.
Table 4.1 Average water contact angles on the surface of heated and UV and ozone treated
polystyrene and PMMA.
No Heat
90
95
Average
100
No Heat
90
95
Standard
Deviation
100
Polystyrene PMMA
42.0
36.2
47.3
59.5
45.8
69.8
47.1
66.4
7.0
4.1
2.3
3.2
2.9
5.4
3.4
4.4
Table 4.2 Average carboxylic acid surface concentration on the surface of heated and UV and
ozone treated polystyrene and PMMA.
No Heat
90
95
Average
100
No Heat
90
95
Standard
Deviation
100
Polystyrene PMMA
11.8
9.8
6.7
9.9
7.2
9.8
6.8
9.7
1.71
0.10
0.94
0.36
0.96
0.15
0.82
0.11
61
Table 4.3: Average Water Contact Angles for UV and Ozone Treated Polystyrene samples stored
in various buffers at acidic, neutral, or basic pH.
Average
Standard
Deviation
5
6
7
8
9
55.9
53.9
Water 55.2
57.1
59.1
61.7
PBS
57.2 60.0 62.4
HEPES
Sodium
59.8 64.9 61.8
Borate
52.6 54.0 55.6
Tris
4.2
3.6
5.2
Water
2.8
3.8
4.7
PBS
4.9 5.71 5.9
HEPES
Sodium
5.3 4.6 3.4
Borate
4.5 4.5 5.0
Tris
Table 4.4: Average carboxylic acid surface concentration for UV and Ozone Treated
Polystyrene samples stored in various buffers at acidic, neutral, or basic pH.
Average
Standard
Deviation
Water
PBS
HEPES
Sodium
Borate
Tris
Water
PBS
HEPES
Sodium
Borate
Tris
5
13.5
10.7
6
7
15.4
7.2
6.1 5.0
8
9
9.0
7.1
5.9
6.0
5.1
6.1 6.6
7.8 6.3
3.26
0.48
1.65
0.55
1.43
1.28
1.49 0.26 0.52
0.49 0.97 1.32
0.43 0.20 1.33
62
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