Surface Functionalization of Piezoelectric Aluminum Nitride with Selected Amino Acid and Peptides

124 Pages·2015·8.28 MB·English

Surface Functionalization of Piezoelectric Aluminum
Nitride with Selected Amino Acid and Peptides
by
Edmund Ho Man Chan
A thesis submitted in conformity with the requirements
for the degree of Master of Applied Science
Institute of Biomaterials and Biomedical Engineering
University of Toronto
© Copyright by Edmund Ho Man Chan 2015
Surface Functionalization of Piezoelectric Aluminum Nitride
with Selected Amino Acid and Peptides
Edmund Ho Man Chan
Master of Applied Science
Institute of Biomaterials and Biomedical Engineering
University of Toronto
2015

Abstract
In the present contribution, we elaborate on the covalent attachment of the amino acid
cysteine and selected cysteine-bearing peptides, in aqueous buffered media, onto AlN
surfaces modified with adlayers of one of our homemade bifunctional
alkyltrichlorosilane cross-linking molecules bearing the benzenethiosulfonate head
group. Surface characterizations confirmed the successful covalent immobilization of
cysteine in buffered media, whereas the attachment of the peptides proved to be
difficult as the undesired partial destruction of the adlayer on AlN by hydrolysis in
aqueous/buffered solvent systems, which was confirmed in a separate study, appeared
to have interfered with the covalent attachment and resulted in one of the peptides
failing to immobilize. Future directions from this will focus on optimizing the solvent
conditions for the cysteine/peptide immobilizations and the implementation of the
surface chemistry to the covalent functionalization of AlN with biologically significant

protein fragments, among them the antigen-binding fragment of antibodies.

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Acknowledgments

Professor Michael Thompson, my thesis supervisor, thanks for your guidance, support, and
friendship in my past few years in graduate school, as I would not have been where I am right
now without you believing in my potential as a researcher.

Dr. Alexander Romaschin, from St. Michael’s Hospital, Dr. Kagan Kerman, from University of
Toronto Scarborough, and Professor Ulrich Krull, from University of Toronto Mississauga,
thanks for being on my supervisory/examination committee and looking over my thesis and
giving suggestions.

Dr. Paul Galvin and Dr. Nathan Jackson, from Tyndall National Institute (Cork, Ireland), I am
especially grateful to the two of you for preparing the aluminum nitride films much needed for
my research as well as sharing with me the same research interests in understanding the
surface chemistry on aluminum nitride.

Dr. Christophe Blaszykowski, thanks for synthesizing the molecules I much needed for my
project, as my research thesis would not have been possible without them. As well, I am
grateful to you especially for making the time out of your busy work schedule to discuss with
me with respect to the possible experiments which would prove important for my thesis, to
provide feedback on my research data, and to give me suggestions on next steps with regards
to my research.

Dr. Sonia Sheikh, Pasquale “Pat” Benvenuto, Miguel Neves, Elaine Chak, Brian de la
Franier, Niall Crawley, Jack Sheng, Kiril Fedorov, Natalia Pawlowska, Ellie Wong, Tairan
Yang, Rúben Machado, Jenise Chen, Tyrally Ordinario, and many other past and present
colleagues in the Thompson research group, thanks for all your friendship, caring, and support
in my past couple of years in graduate school.

Dr. Peter Brodersen, from Surface-Interface Ontario, thanks for running all the X-ray
photoelectron spectroscopy sample characterizations in a timely fashion whenever I asked, as
well as showing me how to process and interpret the XPS spectra of the samples. These XPS
characterizations prove to be invaluable and extremely useful for my research thesis.
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Here, I also like to thank the Natural Sciences and Engineering Research Council of
Canada (NSERC), University of Toronto, and especially Institute of Biomaterials and
Biomedical Engineering (IBBME) for funding for my last couple of years of graduate school.

Chapman Lau, my dear friend, and many others whom I am close to in the Christian fellowship
Nexis Community from Richmond Hill Christian Community Church (RHCCC), thanks for all
your friendship, love, and care in my walk in life, especially in these past years during my
Masters program. You guys are part of the reason I am able to succeed as a graduate student
and make a step forward in scientific research.

Mom (Tammy Chan) and Dad (Joe Chan), thanks for taking care of my daily needs while I am
constantly pre-occupied with my graduate school work, so that I am able to focus on pursuing
my education and equipping myself with plentiful research experience for my scientific research
career.

Above all, God Almighty, My Heavenly Father, thanks for giving me the opportunity to pursue
graduate studies. I am grateful to You for blessing me in my last couple of years in graduate
school, especially by putting the all the above mentioned people around me, and many others,
for guidance at the much needed moments during my project.

I dedicate my thesis to all of you.

Edmund Ho Man Chan
June 2015

“Trust in the LORD with all your heart and lean not on your own understanding;
in all your ways submit to him, and he will make your paths straight.”

(Proverbs 3:5-6, NIV)

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Table of Contents

Abstract .................................................................................................................................. ii
Acknowledgments ................................................................................................................ iii
Table of Contents ................................................................................................................... v
Table of Figures ................................................................................................................... vii
List of Appendices ................................................................................................................ xi
List of Abbreviations ........................................................................................................... xii
1 Introduction ..................................................................................................................... 1
1.1 Biosensors ...................................................................................................... 1
1.2 The Piezoelectric Effect and Acoustic Wave Devices ...................................... 3
1.3 Quartz-based biosensors ................................................................................ 4
1.3.1 Thickness Shear Mode (TSM) Device ............................................................. 4
1.3.2 Electromagnetic Piezoelectric Acoustic Wave Sensor (EMPAS) ..................... 7
1.3.3 Wireless-electrodeless quartz crystal microbalance (WE-QCM) ...................... 9
1.4 Replacing quartz with other piezoelectric materials as transducer................. 10
1.5 Aluminum Nitride: An alternative piezoelectric material to quartz .................. 11
1.6 Recently reported AlN BAW sensors: The AlN FBAR device ........................ 13
1.7 The need for an electrode-free AlN BAW biosensor detection platform ......... 13
1.8 Surface modifications of AlN with organosilane adlayers .............................. 14
1.8.1 Recently reported studies on organosilane surface chemistry on AlN ........... 15
1.8.2 Our approach: Bifunctional Organotrichlorosilane Adlayers .......................... 16
1.8.2.1 Mechanism of Organotrichlorosilane Adlayer Formation ................................ 18
1.8.2.2 Covalent Immobilization of Biological/Chemical Probes onto Organo-
trichlorosilane-modified Surfaces via Reaction with Terminal Group ............. 20
1.8.2.3 The benzenethiosulfonate-based alkyltrichlorosilane linker ........................... 21
1.8.2.4 Recent advance: Surface Modification of AlN with TUBTS Adlayer and
Subsequent Immobilization of Thiolated Biotin Probes .................................. 23
1.8.2.5 Other Potential Probes: Amino Acid and Peptides ......................................... 24
1.9 Thesis Project ............................................................................................... 26
1.9.1 Research Objectives and Description of Surface Treatments ........................ 26
1.9.2 X-Ray-Photoelectron Spectroscopy .............................................................. 28
2 Materials and Methods ................................................................................................. 33
2.1 General Remarks .......................................................................................... 33
2.2 AlN Film Deposition Methods ........................................................................ 34
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2.3 Surface oxidation/Hydroxylation of AlN films by ozone (and subsequent
water physisorption onto oxidized AlN films for TUBTS adlayer
formation) ...................................................................................................... 35
2.4 TUBTS Adlayer Formation on oxidized/hydroxylated AlN films ..................... 36
2.5 L-Cysteine immobilization onto TUBTS-modified AlN films............................ 38
2.6 c(RGDfC) peptide immobilization onto TUBTS-modified AlN films ................ 39
2.7 GRGDSPC peptide immobilization onto TUBTS-modified AlN films .............. 40
2.8 CDPGYIGSR peptide immobilization onto TUBTS-modified AlN ................... 41
2.9 Exposure of TUBTS-modified AlN films to aqueous/buffered
environment for test of stability to cleavage ................................................... 42
2.10 XPS Surface Characterization ....................................................................... 43
3 Results and Discussion ................................................................................................ 44
3.1 Covalent Immobilization of Cysteine on TUBTS-treated AlN films ................. 44
3.2 Covalent Functionalization of TUBTS-treated AlN films with peptides ........... 58
3.3 Susceptibility of TUBTS adlayer on AlN films to hydrolytic cleavage ............. 73
4 Conclusion .................................................................................................................... 84
5 Next Steps and Future Work ........................................................................................ 85
5.1 Determining maximum incubation time of organtrichlorosilane adlayers
on AlN films in aqueous environments before detectable hydrolytic
cleavage ....................................................................................................... 85
5.2 Different solvent environments for cysteine and peptide immobilization ........ 86
5.3 Control Experiment: Exposure of cysteine and peptide adsorption
exposed to AlN in the absence of TUBTS adlayers ....................................... 86
5.4 Another potential probe: The antibody antigen-binding fragment .................. 87
6 Final Remarks ............................................................................................................... 88
References ........................................................................................................................... 89
Appendices .......................................................................................................................... 95

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Table of Figures

Figure 1.1 Components of a biosensor ......................................................................... 2
Figure 1.2 The thickness-shear-mode (TSM) device .................................................... 5
Figure 1.3 A visualization of the thickness shear mode (TSM) wave propagating
through the quartz disc ................................................................................ 5
Figure 1.4 Schematic of the electromagnetic piezoelectric acoustic sensor
(EMPAS) ......................................................................................................... 8
Figure 1.5 Structure of the wireless-electrodeless quartz crystal microbalance
(WE-QCM) ...................................................................................................... 9
Figure 1.6 Crystal lattice structure of AlN: The hexagonal (wurtzite) phase (Left)
and the distorted tetrahedral structure (Right) ......................................... 11
Figure 1.7 Comparison of selected piezoelectric properties between aluminum
nitride and quartz ........................................................................................ 12
Figure 1.8 Surface functionalization of inorganic substrates with organosilane
adlayers ....................................................................................................... 14
Figure 1.9 Overall structure of the bifunctional organotrichlorosilane linkers ........ 16
Figure 1.10 Selection of functionalizable head groups(benzenethiosulfonate, 2,2,2-
trifluoroethyl ester, pentafluorophenyl ester, and 2-chloroacetate)
explored in our laboratory in our development of the bifunctional
organotrichlorosilane linkers ..................................................................... 17
Figure 1.11 Mechanism of organotrichlorosilane adlayer formation .......................... 19
Figure 1.12 Structure of the benzenethiosulfonate bearing alkyltrichlorosilane
linker (TUBTS) developed in our laboratory. [Name: S-(11-
trichlorosilyl)undecanylbenzenthiosulfonate] .......................................... 21
Figure 1.13 Reaction of thiosulfonates with sulfhydryl groups (thiols) to generate
disulfide bonds ........................................................................................... 21
Figure 1.14 Potential attachment of thiolated (sulfhydryl-bearing) probe molecule
onto inorganic substrates modified with the TUBTS adlayer .................. 22
Figure 1.15 TUBTS adlayer formation on AlN and subsequent covalent
immobilization of thiolated biotin probes, the latest example of AlN
surface modification carried out in our laboratory ................................... 23
Figure 1.16 Generic structure of an amino acid (L and D isomers) ............................ 24
Figure 1.17 Structures of the 20 naturally occurring amino acids (L-isomer) ............ 24
Figure 1.18 Sample structure of a peptide (RGD peptide example) ............................ 25
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Figure 1.19 Covalent attachment of cysteine and its residues onto TUBTS-
modified AlN films ...................................................................................... 25
Figure 1.20 Cysteine-bearing peptides to be tested for covalent attachment onto
TUBTS adlayers on AlN films (TUBTS-reactive cysteine residue
highlighted blue) ......................................................................................... 27
Figure 1.21 X-Ray photoelectron spectroscopy instrumentation ............................... 29
Figure 1.22 The photoelectric effect, wherein an X-ray photon ejects an electron
from a core level (1s in this example) due to the X-ray energy (hv)
being larger than the binding energy of the core electron ....................... 29
Figure 1.23 Angle-resolved X-Ray photoelectron spectroscopy (ARXPS) ................. 31
Figure 2.1 AlN surface oxidation and subsequent water physisorption ................... 35
Figure 2.2 TUBTS Adlayer Formation onto AlN films ................................................. 36
Figure 2.3 Covalent immobilization of cysteine onto TUBTS-modified AlN films .... 38
Figure 2.4 Covalent immobilization of c(RGDfC) peptide onto TUBTS-modified
AlN films ...................................................................................................... 39
Figure 2.5 Covalent immobilization of GRGDSPC peptide onto TUBTS-modified
AlN films ...................................................................................................... 40
Figure 2.6 Covalent immobilization of CDPGYIGSR peptide onto TUBTS-modified
AlN films ...................................................................................................... 41
Figure 2.7 Potential removal of TUBTS adlayer on AlN due to hydrolysis in
aqueous/buffered environment .................................................................. 42
Figure 2.8 Literature Al 2p, S 2p, and N 1s XPS data relevant for the executed AlN
surface treatments ...................................................................................... 43
Figure 3.1 Relevant surface structures of treated AlN films for covalent
immobilization of cysteine: Untreated (AlN-X), oxidized (AlN-OH),
TUBTS-modified (AlN-TUBTS), and cysteine-treated (AlN-CYS) ............. 45
Figure 3.2 Surface-sensitive and bulk-sensitive Al 2p XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), and
cysteine-treated (AlN-CYS) AlN surfaces .................................................. 46
Figure 3.3 Surface-sensitive and bulk-sensitive Si 2p XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), and
cysteine-treated (AlN-CYS) AlN surfaces .................................................. 47
Figure 3.4 Surface-sensitive and bulk-sensitive S 2p XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), and
cysteine-treated (AlN-CYS) AlN surfaces .................................................. 48
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Figure 3.5 Surface-sensitive and bulk-sensitive C 1s XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), and
cysteine-treated (AlN-CYS) AlN surfaces .................................................. 49
Figure 3.6 Surface-sensitive and bulk-sensitive N 1s XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), and
cysteine-treated (AlN-CYS) AlN surfaces .................................................. 50
Figure 3.7 Surface-sensitive and bulk-sensitive O 1s XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), and
cysteine-treated (AlN-CYS) AlN surfaces .................................................. 51
Figure 3.8 Element-to-element ratios of TUBTS-modified (AlN-TUBTS) and
cysteine functionalized (AlN-CYS) AlN surfaces ...................................... 56
Figure 3.9 Relevant surface structures of treated AlN films for covalent
immobilization of cysteine-bearing peptides ........................................... 59
Figure 3.10 Relevant surface structures of treated AlN films for covalent
immobilization of cysteine-bearing peptides (continued) ........................ 60
Figure 3.11 Surface-sensitive and bulk-sensitive Al 2p XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), c(RGDfC)-
treated (AlN-c(RGDfC)), GRGDSPC-treated (AlN-GRGDSPC), and
CDPGYIGSR-treated (AlN-CDPGYIGSR) AlN films. .................................. 61
Figure 3.12 Surface-sensitive and bulk-sensitive Si 2p XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), c(RGDfC)-
treated (AlN-c(RGDfC)), GRGDSPC-treated (AlN-GRGDSPC), and
CDPGYIGSR-treated (AlN-CDPGYIGSR) AlN films. .................................. 62
Figure 3.13 Surface-sensitive and bulk-sensitive S 2p XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), c(RGDfC)-
treated (AlN-c(RGDfC)), GRGDSPC-treated (AlN-GRGDSPC), and
CDPGYIGSR-treated (AlN-CDPGYIGSR) AlN films. .................................. 63
Figure 3.14 Surface-sensitive and bulk-sensitive C 1s XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), c(RGDfC)-
treated (AlN-c(RGDfC)), GRGDSPC-treated (AlN-GRGDSPC), and
CDPGYIGSR-treated (AlN-CDPGYIGSR) AlN films. .................................. 64
Figure 3.15 Surface-sensitive and bulk-sensitive N1s XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), c(RGDfC)-
treated (AlN-c(RGDfC)), GRGDSPC-treated (AlN-GRGDSPC), and
CDPGYIGSR-treated (AlN-CDPGYIGSR) AlN films. .................................. 65
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Figure 3.16 Surface-sensitive and bulk-sensitive O 1s XPS Profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), c(RGDfC)-
treated (AlN-c(RGDfC)), GRGDSPC-treated (AlN-GRGDSPC), and
CDPGYIGSR-treated (AlN-CDPGYIGSR) AlN films. .................................. 66
Figure 3.17 Element-to-element ratio of TUBTS-modified (AlN-TUBTS), c(RGDfC)-
treated (AlN-(RGDfC)), GRGDSPC-treated (AlN-GRGDSPC), and
CDPGYIGSR-treated (AlN-CDPGYIGSR) AlN films ................................... 70
Figure 3.18 Relevant surface structures of treated AlN films for hydrolytic stability
test ............................................................................................................... 74
Figure 3.19 Surface-sensitive and bulk-sensitive Al 2p XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), and PBS-
treated AlN films (AlN-TUBTS*) .................................................................. 75
Figure 3.20 Surface-sensitive and bulk-sensitive Si 2p XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), and PBS-
treated AlN films (AlN-TUBTS*) .................................................................. 76
Figure 3.21 Surface-sensitive and bulk-sensitive S 2p XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), and PBS-
treated AlN films (AlN-TUBTS*) .................................................................. 77
Figure 3.22 Surface-sensitive and bulk-sensitive C 1s XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), and PBS-
treated AlN films (AlN-TUBTS*) .................................................................. 78
Figure 3.23 Surface-sensitive and bulk-sensitive N 1s XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), and PBS-
treated AlN films (AlN-TUBTS*) .................................................................. 79
Figure 3.24 Surface-sensitive and bulk-sensitive O 1s XPS profile of untreated
(AlN-X), oxidized (AlN-OH), TUBTS-modified (AlN-TUBTS), and PBS-
treated AlN films (AlN-TUBTS*) .................................................................. 80

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