controlled vapor deposition of azide-terminated siloxane monolayers

CONTROLLED VAPOR DEPOSITION OF AZIDE-TERMINATED SILOXANE
MONOLAYERS: A PLATFORM FOR TAILORING OXIDE SURFACES
A DISSERTATION
SUBMITTED TO THE DEPARTMENT OF CHEMICAL ENGINEERING
AND THE COMMITTEE ON GRADUATE STUDIES
OF STANFORD UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
Randall Dewayne Lowe, Jr.
August 2011

© 2011 by Randall Dewayne Lowe, Jr. All Rights Reserved.
Re-distributed by Stanford University under license with the author.
This work is licensed under a Creative Commons Attribution-
Noncommercial 3.0 United States License.
http://creativecommons.org/licenses/by-nc/3.0/us/
This dissertation is online at: http://purl.stanford.edu/pp613jc4816
ii

I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Zhenan Bao, Primary Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Christopher Chidsey, Primary Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Approved for the Stanford University Committee on Graduate Studies.
Curtis Frank
Patricia J. Gumport, Vice Provost Graduate Education
This signature page was generated electronically upon submission of this dissertation in
electronic format. An original signed hard copy of the signature page is on file in
University Archives.
iii

Abstract
The controlled deposition of mixed, azide-terminated siloxane monolayers
onto oxide surfaces provides a platform for the covalent attachment of alkyne-
terminated species such as oligonucleotides using the copper-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction.
A convenient method for the vapor deposition of dense siloxane monolayers
onto oxide surfaces was developed. A selectively deuterated silane, (CD3O)3-Si-
(CH2)13-CH3, was synthesized and then vapor deposited in the presence of controlled
amounts of water vapor from the in situ dehydration of MgSO4·7H2O at 110°C.
Monolayer densification was characterized using ellipsometry, Fourier transform
infrared (FTIR) spectroscopy, water contact angle, and electrochemical capacitance
measurements. The CD3 stretching mode of the selectively deuterated silane was
monitored using FTIR spectroscopy to probe the hydrolysis of methoxy groups on
adsorbed silanes while varying the deposition time, amount of water, and amount of
silane. The formation of dense, completely hydrolyzed monolayers required excess
amounts of silane and water reactants.
The optimized vapor deposition conditions were used to form mixed, azide-
terminated monolayers. Dilution of the azide groups with unreactive methyl groups
was achieved from the co-deposition of two silanes. Enrichment of the monolayers
with the higher vapor pressure silane reactant was observed as measured using FTIR
spectroscopy, XPS, and water contact angle goniometry. The enrichment was
attributed to fractionation of the silane vapor in contact with the surface from the
liquid silane source. Quantitative CuAAC reactions of the azide groups in mixed
iv

monolayers were demonstrated with azide surface compositions up to approximately
40%, which suggested that the components in the monolayers were uniformly mixed.
Steric hindrance presumably prevented the quantitative reaction of a purely azide-
terminated monolayer.
Alkyne-terminated oligonucleotides were covalently attached to azide-
terminated monolayers on glass followed by hybridization with complementary
oligonucleotides. However, the azide-terminated monolayers were susceptible to the
nonspecific adsorption of oligonucleotides even after thorough rinsing in aqueous
detergents and buffers. A two-step reaction sequence was developed that involved one
CuAAC reaction to attach oligonucleotides at low density followed by a second
CuAAC reaction with ethynyl phosphonic acid to make the surface more resistant to
nonspecific adsorption. Bulk and single molecule fluorescence measurements were
used to characterize the glass surfaces before and after exposure to dye-labeled
oligonucleotides.
v

Acknowledgements
I first thank my thesis advisor, Professor Christopher E. D. Chidsey, for his
patience and guidance in training me as a scientist and engineer over the past five
years. I have learned a great deal from Prof. Chidsey but am most thankful for his
teaching me how to approach scientific problems and how to effectively communicate
the results of my research. I appreciate Professor Chidsey‟s entrusting me with the role
of laboratory safety coordinator and his allowing me to mentor undergraduate students
and local high school teachers. I also thank Professor Zhenan Bao and Professor Curt
Frank for acting as readers on my thesis committee and for the experience from having
performed research rotations in both of their laboratories during my first year of
graduate school. I thank Professor Stacey Bent for acting as the non-reader on my
committee and for the experience that I received during a teaching assistantship for an
undergraduate laboratory course that she instructed. I thank Professor Juan Santiago
for acting as the chair of my committee and for the experience I gained while
collaborating with one of his former students, Dr. Cullen Buie.
I thank Dr. Tim Harris and Dr. Erin Artin for their excitement and hard work
during our collaboration with Helicos BioSciences. I am thankful for having had the
privilege of working with and learning from both Dr. Harris and Dr. Artin during the
early years of my graduate career. I also thank Helicos BioSciences for funding.
I thank Dr. Todd Eberspacher for his assistance and advice over the years. Dr.
Eberspacher was particularly helpful in resolving safety issues in the laboratory. I
thank the past and current members of the Chidsey group with whom I have had the
privilege of working over the years: Jonathan Prange, Ali Hosseini, Josh Ratchford,
vi

Charles McCrory, Anando Devadoss, Neal Devaraj, Alex Neuhausen, Jacob Woodruff,
Sujatha Raghu, David Lapham, Ryan Vu, and Jim Harriss. I also thank members from
the Stack research group: Matthew Pellow, Pratik Verma, Brian Smith, Eric
Stenehjem, Andrew Thomas, Tim Storr, and Bolin Lin.
I have learned very much from performing research and taking classes while in
graduate school, but I have also learned as much or more from the many great friends I
have made during this time. I thank each of them for their friendship and time. I look
forward to carrying our friendships into the future.
My family deserves special recognition and thanks for their continuous love
and support throughout my life. You have each helped me so much, and I feel there is
no way that I can ever repay you. I thank Shoko for her love and commitment while
standing by me for so many years.
vii

Table of Contents
Abstract………………………….…………………………………………………….iv
Acknowledgements………………………..………………………………………......vi
Table of Contents……………………………..……………………………….…......viii
List of Tables……………………………..……………………………………..….....xi
List of Figures…………..………………..…………………………………….…......xii
Chapter 1: Introduction………………………………………………………………...1
Thesis objective……………….………………………………………………..1
Background…………………………………………………………………….2
Analytical Techniques………………………………………………………...10
Collaborations…………….…………………….…………………………….15
References..…………………………………………………………………...18
Chapter 2: Deposition of Dense Siloxane Monolayers from Water and
Trimethoxyorganosilane Vapor……………………..……………………………......26
Preface………………………………………………………………………...26
Abstract……………………………………………………………………….27
Introduction…………………………………………………………………...28
Experimental...………………………………………………………………..30
Results………………………………………………………………………...35
Discussion.……………………………………………………………………42
Conclusions...…………………………………………………………………47
Acknowledgements……………………………………….…………………..48
Supporting Information………………………………………………….........49
viii

References…………………………………………………………………….56
Chapter 3: Vapor Deposition of Mixed, Azide-Terminated Siloxane Monolayers: A
Modular Strategy for Modifying Oxide Surfaces..…………………………………...63
Preface………………………………………………………………………...63
Abstract……………………………………………………………………….64
Introduction…………………………………………………………………...65
Experimental………………………………………………………………….69
Results………………………………………………………………………...72
Discussion…………………………………………………………………….85
Conclusions…………………………...………………………………………89
Acknowledgements…………………………………………………………...90
Supporting Information………….……………………………………………91
References…..……………………………………………………………..….93
Chapter 4: Attachment and Hybridization of Oligonucleotides on Azide-Terminated
Siloxane Monolayers with Resistance to Nonspecific Adsorption.………………......99
Preface………………………………………………………………………...99
Abstract….…………………………………………………………………..100
Introduction………………………………………………………………….101
Experimental………………………………………………………………...105
Results and Discussion………………………………………………...……109
Conclusions………………………………………………………………….120
Acknowledgements...………………………………………………………..120
Supporting Information……………………………………………………...121
ix

References……………………………………………………………….......131
x

List of Tables
Chapter 2, Table S1. Calculated CH3OH:H2O Ratio in Deposition Chamber
Assuming Complete Hydrolysis and Condensation…….…………………………….54
Chapter 4, Table S1. Nonspecific Adsorption of Dye-Labeled Mononucleotides onto
Azide-Terminated Monolayers Reacted with Alkyne-Terminated Oligonucleotides and
Propargyl Alcohol during Single Molecule Sequencing by Synthesis……………...129
Chapter 4, Table S2. Nonspecific Adsorption of Dye-Labeled Mononucleotides onto
Epoxide-Terminated Monolayers Reacted with Amine-Terminated Oligonucleotides
and Phosphate during Single Molecule Sequencing by Synthesis ……………..…...130
xi

List of Figures
Chapter 1, Figure 1. Schematic of the platform developed in this thesis to tailor the
properties of oxide surfaces for the covalent attachment and hybridization of
oligonucleotides with minimal nonspecific adsorption..……………..………………..3
Chapter 1, Figure 2. Chemical structures of two trimethoxysilane adsorbates used in
this thesis.……………..………………………………………………………………..6
Chapter 2, Figure 1. Schematic of the glass vacuum chamber that was used for
siloxane monolayer vapor depositions onto oxide surfaces from liquid silane and
MgSO4·7H2O reactants…………………………………...…………..………………32
Chapter 2, Figure 2. Time dependence of 100 µL (CD3O)3-Si-(CH2)13-CH3
monolayer vapor deposition onto silicon oxide without and with 0.5 g MgSO4·7H2O
present …………………………………...…………..…………………….…………36
Chapter 2, Figure 3. Carbon-deuterium and carbon-hydrogen stretching regions of
the p-polarized Brewster‟s angle transmission FTIR spectra of monolayers vapor-
deposited onto silicon oxide for 12 h from 100 µL (CD3O)3-Si-(CH2)13-CH3 without
and with 0.5 g MgSO4·7H2O present….....…………..…………………….…………37
xii

Chapter 2, Figure 4. Plot of the ellipsometric thickness and the integrated FTIR
symmetric CD3 stretching absorbance versus mass of MgSO4·7H2O added for 12 h
depositions onto silicon oxide with various initial volumes of (CD3O)3-Si-(CH2)13-
CH3. ….....…………..…………………………………………..………….…………39
Chapter 2, Figure 5. Plot of the electrochemical capacitance versus scan rate during
cyclic voltammetry after 12 h depositions of 100 µL (CD3O)3-Si-(CH2)13-CH3 onto
ITO electrodes without and with 0.5 g MgSO4·7H2O present.....………….…………41
Chapter 2, Figure S1. Attenuated total reflectance (ATR) FTIR spectrum of the bulk,
liquid tetradecyl-tri(deuteromethoxy)silane used for siloxane monolayer vapor
depositions………………………………………………….......………….…………49
Chapter 2, Figure S2. NMR spectrum of tetradecyl-tri(deuteromethoxy)silane that
was synthesized and used for siloxane monolayer vapor depositions….….…………50
Chapter 2, Figure S3. Plot of the current versus applied potential measured during
cyclic voltammetry for a siloxane monolayer on ITO exposed to 0.1 M NaClO4 in
water at a scan rate of 1000 mV/s…………………………………...….….…………51
xiii

Chapter 2, Figure S4. Carbon-hydrogen stretching region of the p-polarized
Brewster‟s angle transmission FTIR spectra of siloxane monolayers on silicon oxide
after one 12 h vapor deposition and two repeated 12 h vapor depositions...…………52
Chapter 2, Figure S5. Time dependence of the dehydration of MgSO4·7H2O at
110°C under reduced pressure starting with 0.5 g MgSO4·7H2O…..……...…………53
Chapter 3, Figure 1. Modular strategy for the covalent modification of oxide surfaces
using mixed, azide-terminated siloxane monolayers followed by the Cu-catalyzed
azide-alkyne cycloaddition (CuAAC) reaction with terminal alkynes to attach species
of interest….………………………………………………………….…...…..………68
Chapter 3, Figure 2. Azide asymmetric and carbon-hydrogen stretching regions of
the p-polarized Brewster‟s angle transmission FTIR spectrum of a χ N3 ,liquid = 1.0
monolayer on silicon oxide.………………………………………….…...…..………73
Chapter 3, Figure 3. High-resolution Si 2p and N 1s XPS spectra for a χ N3 ,liquid = 1.0
monolayer on silicon oxide after 12 h of vapor deposition from 100 µL of 11-
azidoundecyltrimethoxysilane with 0.5 g MgSO4·7H2O present.……………………74
xiv

Chapter 3, Figure 4. Time dependence of vapor deposition of χ N3 ,liquid = 1.0
monolayers onto silicon oxide surfaces from 100 µL of 11-
azidoundecyltrimethoxysilane with and without 0.5 g MgSO4·7H2O present…….…77
Chapter 3, Figure 5. Carbon-fluorine and azide asymmetric stretching regions of the
p-polarized Brewster‟s angle transmission FTIR spectrum of (a) χ N3 ,liquid = 1.0 and (b)
χ N3 ,liquid = 0.75 monolayers on silicon oxide before and after 1 h of a CuAAC reaction
with 4-ethynyl-α,α,α-trifluorotoluene………………………………………..…….…79
Chapter 3, Figure 6. Relative surface compositions of mixed monolayers vapor
deposited from the azidosilane and alkyltrimethoxysilane diluents.………..…….…82
Chapter 3, Figure 7. High-resolution XPS spectra of the C 1s, N 1s, and F 1s
photoelectron regions for a χ N3 ,liquid = 0.75 monolayer on silicon oxide before and after
1 h of a CuAAC reaction with 4-ethynyl-α,α,α-trifluorotoluene..……….…..…….…85
Chapter 3, Figure S1. NMR spectrum of the synthesized 11-
azidoundecyltrimethoxysilane used for siloxane monolayer vapor depositions.......…91
xv

Chapter 3, Figure S2. Chemical structure drawing of TTMA, the copper ligand used
for CuAAC reactions…………………………………………………………........…92
Chapter 4, Figure 1. Schematic of the platform developed for the attaching and
hybridizing dye-labeled oligonucleotides on oxide surfaces.…….……………....…104
Chapter 4, Figure 2. Cy3 fluorescence emission spectra of vapor-deposited azide-
terminated monolayers on glass excited at 530 nm before and after exposure to 10 nM
Cy3-RG2-amine …………………………………………………….…………....…111
Chapter 4, Figure 3. Cy3 bulk fluorescence emission spectra and single molecule
fluorescence images of vapor-deposited azide-terminated monolayers on glass before
and after a 1 h exposure to Cy3-RG2-alkyne with and without the Cu(I)
catalyst………………...…………………………………………….…………....…114
Chapter 4, Figure 4. Cy5 fluorescence emission spectra of azide-terminated
monolayers on glass exposed to hybridization conditions for Cy5-labeled
oligonucleotides.……...…………….……………………………….…………....…117
Chapter 4, Figure S1. Reaction scheme for the synthesis of the alkyne-terminated
oligonucleotide that was used in this study for CuAAC reactions with azides.….…121
xvi

Chapter 4, Figure S2. Cy3 fluorescence emission spectrum of 10 nM of a Cy3-
labeled oligonucleotide, Cy3-RG2-amine, in water excited at 530 nm. Cy3
fluorescence emission spectra of vapor-deposited azide-terminated monolayers on
glass before and after exposure to 10 nM Cy3-alkyne with and without the Cu(I)
catalyst for the CuAAC reaction………………………………………………….…123
Chapter 4, Figure S3. Chemical structure of the alkyne-terminated Cy3 dye (Cy3-
alkyne) that was covalently attached to azide-terminated monolayers using the
CuAAC reaction.………………………………………………………………….…124
Chapter 4, Figure S4. High-resolution XPS spectra of the P 2p and N 1s
photoelectron regions for an azide-terminated monolayer on silicon oxide before and
after a CuAAC reaction with 10 mM ethynyl phosphonic acid…………....…….…125
Chapter 4, Figure S5. Fluorescence emission spectra of azide-terminated monolayers
on glass before and after a 1 h exposure to 10 nM of the Cy3-RG2-alkyne without and
with Cu(I) present followed by propiolic acid CuAAC…………………....…….…127
xvii

Chapter 1: Introduction
Thesis objective
The objective of my thesis is to develop a platform for tailoring the properties
of oxide surfaces. I developed a method for the controlled deposition of self-
assembled monolayers presenting azide groups at tunable density onto oxide surfaces
from trimethoxysilane and water vapor. The azide groups on the surface can react with
terminal alkynes using the Cu-catalyzed azide-alkyne cycloaddition reaction to
covalently attach species to tailor the surface properties. I applied this general platform
to the immobilization of oligonucleotides onto glass surfaces and their subsequent
hybridization with complementary oligonucleotides while resisting nonspecific
adsorption. This platform may enable the further development of complex surfaces for
biosensing and other applications.
1

Background
Glass surfaces are interesting substrates for the surface immobilization of
species of interest because they are relatively cheap, unreactive to many chemicals,
stable at elevated temperature, optically transparent, and they exhibit low background
fluorescence. 1,2,3 Some technologies rely on chemistry or measurements that are most
easily performed at fixed sites on surfaces. For example, many biosensors are based on
the immobilization of bioanalytical probes such as oligonucleotides 4 and proteins 5 to
surfaces such as glass. The work in this thesis was originally motivated by a
collaboration with Helicos BioSciences, a company commercializing a next-
generation, single molecule DNA sequencing by synthesis technology on glass
surfaces. 6,7
My goal in the collaboration was to produce a robust platform on glass
surfaces for the covalent attachment of oligonucleotide targets at controllable density
that could undergo hybridization and capture complementary oligonucleotides with
minimal nonspecific adsorption. The inherently low background fluorescence of the
high quality glass substrates needed to be maintained after the surface treatments to be
useful for single molecule fluorescence measurements. The general platform used in
this thesis to tailor the properties of oxide surfaces is shown schematically in Figure 1.
This strategy involves the controlled deposition of mixed siloxane monolayers with
tunable azide densities followed by Cu-catalyzed azide-alkyne cycloaddition
(CuAAC) reactions with terminal alkynes on oligonucleotides and other species to
resist nonspecific adsorption. This platform may be used for the immobilization of
other alkyne-terminated species besides oligonucleotides. The inspiration for this
2

approach comes from the extensive work of Devaraj et al. in which mixed, azide-
terminated monolayers adsorbed from thiols onto gold were found to be excellent
platforms for tailoring electrode surfaces. 8,9,10
1.
2.
Oxide Surface Oxide Surface
Figure 1. Schematic of the platform developed in this thesis to tailor the properties of
oxide surfaces for the covalent attachment and hybridization of oligonucleotides with
minimal nonspecific adsorption. The strategy first involves the controlled deposition
of mixed, azide-terminated siloxane monolayers followed by CuAAC reactions with
alkyne-terminated oligonucleotides and other species (represented by the black
squares) to tune the properties of the surface.
3

Cu-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) Reaction
A key component of the platform is the CuAAC reaction, discovered
independently by Sharpless and Meldal in 2002. 11,12 This cycloaddition reaction
between azide groups and terminal alkyne groups is considered the „cream of the crop‟
of the family of reactions termed „click‟ reactions. 13 The CuAAC reaction is an ideal
candidate for surface attachment because it is selective (tolerant to other functional
groups), quantitative, and proceeds rapidly in aqueous and organic solvents under
ambient atmospheric conditions. The product of the CuAAC reaction is a 1,4-
disubstituted 1,2,3-triazole that is thermally, oxidatively, reductively and
hydrolytically stable. 14 The versatility of the CuAAC reaction has been demonstrated
through the attachment of a variety of alkyne-terminated substituents such as:
oligonucleotides, 10,15,4,16 proteins, 17,18,19 electroactive compounds, 9,20 fluorescent
dyes, 21,22 various polymers, 23,24 and particles 25 to azide-terminated surfaces. The Cu(I)
catalyst is water-soluble and can be maintained in the +1 oxidation state by use of an
appropriate Cu(I)-stabilizing ligand and an appropriate water-soluble reductant such as
ascorbate. Cu-free cycloaddition reactions between highly strained disubstituted
alkynes and azides have recently been discovered for biological applications in which
copper cannot be tolerated, 26 but the utility of this reaction on surfaces is not explored
in this thesis. The CuAAC reaction was chosen over the multitude of other interfacial
chemistries that have been used to couple species to monolayers 27 based on the
characteristics mentioned above.
Before using the CuAAC reaction to attach oligonucleotides and other species
to surfaces, I needed to first prepare azide-terminated surfaces. Azide-terminated
4

surfaces were chosen over alkyne-terminated surfaces because azides can be probed
relatively easily using FTIR spectroscopy and XPS. 8 By decoupling the attachment of
the oligonucleotides from siloxane monolayer formation, I was able to independently
optimize conditions for CuAAC reactions and for the vapor deposition of azide-
terminated monolayers as the only reactive functionality on an otherwise passive glass
or silicon oxide surface. 28
Deposition of Siloxane Monolayers
Siloxane monolayers can be used to tailor the properties and reactivity of
silicon oxide and other oxide surfaces. Since Sagiv et al. systematically studied the
solution deposition of self-assembled siloxane monolayers onto glass surfaces, 29 a
large amount of research has followed to better understand siloxane monolayer
deposition and to use the monolayers for various applications as summarized in more
thorough literature reviews. 30,31,32,28,33,34
Siloxane monolayers are deposited by exposing oxide surfaces to reactants
with silane headgroups for surface attachment and with compatible tailgroups such as
alkyl chains that can promote self-assembly through Van der Waals attractions. 33 The
tailgroups can also be terminated with organic functional groups to be presented on the
surface. These functional groups should not interact strongly or bond with the oxide
surface nor should they react with the silane headgroups to interfere with monolayer
formation. 35 Examples of two silane reactants that are discussed in this thesis are
shown in Figure 2.
5

Figure 2. Chemical structures of two trimethoxysilane adsorbates used in this thesis.
Alkylsilane reactants can be mono, di, or trifunctional depending on the
number of hydrolyzable chloro or alkoxy groups present. 34 The formation of siloxane
monolayers from trimethoxysilanes, such as the ones used in this study, is a stepwise
process. Trimethoxysilanes can react directly with hydroxyl groups on the surface to
form siloxane linkages, or they can undergo hydrolysis reactions with water to
produce hydroxyl groups on the silanes. Methanol is a byproduct of both reactions.
Condensation reactions can then occur between hydroxyl groups on silanes and on the
surface or between hydroxyl groups on neighboring silanes to produce siloxane
linkages and water. As these reactions proceed to completion, a dense siloxane
monolayer is formed that can be described as a two-dimensional network polymer
with occasional siloxane linkages to the oxide surface. 36
Two general methods for depositing siloxane monolayers are solution 29 and
vapor-phase deposition. 37 The vapor deposition of siloxane monolayers was chosen
over solution deposition because vapor depositions often lead to monolayers with less
6

particulate contamination from siloxane oligomers on the surface than solution
depositions as inferred from atomic force microscopy images. 38 This particulate
contamination could interfere with monolayer formation or with subsequent surface
reactions on the monolayer. Variations of solution deposition such as spin-coating
have been used to produce ultrasmooth siloxane monolayers. 39
Hong et al. showed that dense siloxane monolayers similar to those deposited
from solution could be achieved using vapor depositions with the deliberate addition
of water as a reactant. 40 Water not only acts as a reactant during hydrolysis reactions,
but it is also a product of condensation reactions between hydroxyl groups. Overall,
the expected stoichiometric ratio of water to trimethoxysilane during siloxane
monolayer formation is 1.5 to 1 assuming complete hydrolysis and condensation. 41
Therefore, control over the amount of water as a reactant should be important for the
deposition of dense monolayers from trimethoxysilanes in both the solution phase and
the vapor phase. Although vapor depositions have been performed
with 40,42,43,44,45,46,47,48,49 and without 50,51,52,53,54,55,56,57,58 the deliberate addition of water,
the effect of water addition during vapor deposition has not been investigated to the
level of detail as for solution deposition. One approach to studying the effect of water
addition is to monitor the presence of unhydrolyzed chloro or alkoxy groups on
adsorbed silanes under various deposition conditions.
The hydrolysis of chlorosilanes has been monitored using FTIR 59,60 and
XPS 61,62,63,50 to probe for the presence of Si-Cl bonds on oxide surfaces during
monolayer deposition. However, there is not an equivalent method for clearly
monitoring the hydrolysis of alkoxysilanes. For example, the presence of
7

unhydrolyzed ethoxy groups during the solution deposition of monolayers from
aminopropyltriethoxysilane onto silicon oxide was inferred from peaks in FTIR
spectra assigned to ethoxy groups, but these peaks were obscured by other stretching
modes. 64 I sought to provide a clearer method for monitoring methoxy group
hydrolysis on oxide surfaces to study the effect of water addition during vapor
deposition. As discussed in Chapter 2, this was accomplished by synthesizing and
vapor depositing a selectively deuterated silane, (CD3O)3-Si-(CH2)13-CH3, and then
monitoring C-D stretching modes attributable to the deuteromethoxy groups.
Due to the low vapor pressure of alkylsilane reactants, vapor depositions are
usually performed at elevated temperature and under reduced pressure. However,
experimental setups for performing vapor depositions range widely from complex
commercial vacuum systems 65,57 to simpler vessels operated at atmospheric pressure. 55
For simplicity, I used a greaseless, glass vacuum desiccator into which the reactants
and surfaces were placed remotely from each other. This was followed by pumping
and sealing the chamber before heating it in an oven. A schematic of the chamber is
provided in Chapter 2. This method was adapted from Ashurst et al. except their
desiccator chamber was more complex with external reactant feeds. 42 Because there
are no reactant feeds on the deposition chamber used in this study, a water source that
would survive pumping to reduced pressure was needed. This problem was solved by
using the in situ dehydration of MgSO4·7H2O to deliver controlled amounts of water.
Mixed, Azide-Terminated Siloxane Monolayers
Once conditions for vapor depositing dense, methyl-terminated siloxane
monolayers were established, the vapor deposition of mixed, azide-terminated
8

monolayers was studied. Azide-terminated monolayers are often formed by reacting
bromide-terminated monolayers with sodium azide. 66,22 However, the nucleophilic
substitution reaction does not always result in quantitative conversion of the bromides
to azides. This problem was avoided by first synthesizing and then vapor depositing an
azide-terminated silane. Once a dense monolayer of azide groups is formed, steric
hindrance may prevent their quantitative conversion using the CuAAC reaction
depending on the alkyne-terminated species. 8 Therefore, the dilution of the azide
groups with unreactive groups such as methyl groups may be desirable.
Mixed siloxane monolayers are not only attractive for controlling the spacing
between groups on the surface, but they can also be used to add additional complexity
to oxide surfaces for more precise surface engineering. Binary mixed monolayers are
usually formed by the simultaneous co-deposition of two adsorbates from
solution, 67,35,68,69 but they can also be formed by sequential deposition. 70,71,72 I opted to
use the co-deposition of trimethoxysilane adsorbates from the vapor phase to study
mixed monolayer deposition as reported in Chapter 3. The ability to probe the relative
surface compositions of mixed monolayers is desirable because the surface
compositions are often different than the compositions of the initial reactant mixtures
from which they were deposited. 73,69,68,74
Another method for producing mixed monolayers involves the deposition of a
pure or mixed monolayer terminated with reactive functional groups followed by
surface reactions with more than one species. 73,75 This approach to forming mixed
monolayers is used in Chapter 4 to attach oligonucleotides and other species to resist
nonspecific adsorption. Common methods for reducing the nonspecific adsorption of
9

iomolecules include rinsing in buffers and detergents 76 and the addition of functional
groups or molecules known to resist nonspecific adsorption. 77,78,79,80,81,82,83,84 These
functionalities are often hydrophilic in nature and may not be compatible as tailgroup
terminations for controlled siloxane monolayer deposition. The platform developed in
this thesis enables the attachment of functionalities that are incompatible with
controlled monolayer deposition onto oxide surfaces.
Analytical Techniques
Brewster’s Angle Transmission Fourier Transform Infrared (FTIR)
Spectroscopy
FTIR spectroscopy is a surface-sensitive technique that I have used to identify
chemical functional groups on silicon oxide surfaces after various treatments such as
monolayer vapor depositions and CuAAC reactions. FTIR spectra were obtained by
transmitting p-polarized IR light through planar, double-side polished silicon oxide
surfaces that were oriented at the Brewster‟s angle for silicon, 74°, 85 using a custom-
built sample holder. Infrared light is absorbed by samples as vibrational modes are
excited, and the absorbance of the impinging light as a function of its frequency in
wavenumbers is measured with a spectrometer. A deuterated triglycine sulfate
(DTGS) detector was used for all measurements. Chabal et al. popularized the use of
Brewster‟s angle transmission FTIR spectroscopy on silicon surfaces. 86
X-ray Photoelectron Spectroscopy (XPS)
XPS is a surface-sensitive spectroscopy that typically yields elemental
information about a surface. XPS spectra are obtained by irradiating a surface with x-
ray photons in an ultrahigh vacuum chamber. For all spectra in this thesis, a
10

monochromator was used to select Al Kα incident radiation, which has a photon
energy of 1486.6 electron volts (eV). The impinging photons excite electrons from
core energy levels into vacuum where they pass through an analyzer that selects the
electrons based upon their kinetic energies before sending them to a detector.
Although the spectrometer actually measures the kinetic energies of the photoemitted
electrons, spectra are usually plotted in units of electron counts versus binding energy.
The binding energy of the electrons is calculated by subtracting the kinetic energy
measured from the incident photon energy, 1486.6 eV for Al Kα incident radiation.
Survey spectra are obtained by measuring electron counts in the 0-1100 eV
binding energy range, where most elements exhibit one or more photoelectron peaks.
High-resolution XPS spectra are used to more accurately determine binding energies
in order to make inferences about the chemical environment around a particular
element in the sample. For example, the binding energy of an atom will exhibit a
slightly greater binding energy if the atom is bound to a more electron-withdrawing
species. 87
Fluorescence Spectroscopy
Fluorescence spectroscopy was used to detect the presence of fluorescent dyes
on glass surfaces in this thesis. The fluorescent dyes used in this study, Cyanine 3
(Cy3) and Cyanine 5 (Cy5), absorb and emit light in the visible region of the
electromagnetic spectrum. A fluorescent dye can absorb a photon of specific energy
which results in the excitation of an electron from its ground state into an excited
singlet state that may be composed of a number of vibrational states. Once in an
excited state, the dye undergoes vibrational relaxation to the lowest vibrational level of
11

the excited state. Next, the excited dye can relax back to any vibrational level of the
ground electronic state. This results in the fluorescent emission of a photon
corresponding to the energy difference between the two states. Because the electron
relaxes to the lowest vibrational level of the excited state before fluorescing and
returning to the ground state, the emitted light is usually lower in energy than the
excitation light. This phenomenon is known as the Stokes shift and can be measured
by performing excitation and emission scans of the fluorescent dye. 88
Fluorescence spectra were obtained by exposing glass surfaces to excitation
light from a 450 watt Xenon lamp source after passing through a monochromator and
a bandpass filter. Emitted light was collected using front-face detection from the
surface after passing the light through another bandpass filter to reduce stray
excitation light and another set of monochromators before reaching a detector. Front-
face detection of the emitted light was used to reduce the detection of reflected and
scattered excitation light.
Ellipsometry
Ellipsometry is an optical measurement that can be used to determine the
thickness of a thin layer on a surface. 33 A helium-neon laser emits 632.8 nm
unpolarized light that passes through a polarizer to become plane-polarized with the
polarization vector oriented 45° from the sample surface. Plane-polarized light
consists of s- and p-polarized light components, which are characterized by the
orientations of their polarization vectors being either parallel (s) or perpendicular (p)
with respect to the surface. The plane polarized light undergoes a single reflection
from the surface with the s- and p-polarized components being reflected to different
12

extents and with different phase delays. After reflection, the relative phase and
amplitude of the s- and p-components of the light are determined. Ellipsometry
measures two parameters, Ψ and Δ, which are related to the complex refractive indices
of the film and of the substrate. Ψ is the angle, the arctangent of which is the ratio of
the amplitudes of the s- and p-polarized components, and Δ is the shift in the phase
between the s- and p-polarized components before and after reflection from the
surface. For films with thicknesses less than 50 Å, such as the monolayers in this
thesis, the thickness can be calculated from the measured Ψ and Δ values with an
appropriate estimate of the film refractive index. A film refractive index of 1.46 was
used for all calculations in this thesis. The thickness calculation is normally performed
as part of the measurement by the ellipsometry software on the instrument computer.
Water Contact Angle Goniometry
Water contact angle goniometry can be used to probe the relative
hydrophobicity or hydrophilicity of a surface. In practice, a water droplet is dispensed
from a needle in close contact to the surface and the water contact angle between the
solid surface and the bottom of the sessile droplet is quickly measured on both sides of
the droplet using a measuring telescope with a protractor in the eye piece. Young‟s
equation describes the balance of forces between a surface and a water droplet,
cos
(1)
LV
where γLV, γSV, γSL, are the interfacial surface tensions at the liquid-vapor, solid-vapor,
and solid-liquid interfaces, respectively. 33 A contact angle of 180° represents no
interaction with the surface, while a contact angle of 0° indicates spreading of the
liquid.
13
SV
SL

Electrochemical Capacitance Measurements
Electrochemical capacitance measurements were performed to probe the
packing density of the alkyl chains in vapor-deposited monolayers. Since a conductive
substrate is needed to perform this measurement, monolayers were vapor deposited
onto indium tin oxide (ITO) surfaces, a conductive metal oxide. Cyclic voltammetry
was used to estimate the electrochemical capacitance of monolayers vapor deposited
onto ITO. Cyclic voltammetry experiments were conducted in a three-electrode
electrochemical cell in which the exposed area was defined by a cylindrically-bored
Teflon cone pressed onto the surface. The surface was exposed to an electrolyte
solution of 0.1 M NaClO4 in water inside the cone. During cyclic voltammetry, a
potential is applied to the ITO working electrode relative to the Ag/AgCl/saturated
KCl reference electrode while electrical current into the working electrode is
measured. 89 The potential is swept at a scan rate with units of potential (volts) per unit
time (seconds). A potentiostat is used to measure the current while controlling the
potential and scan rate during cyclic voltammetry. The resulting cyclic voltammogram
is typically plotted in units of current density (microamps per unit area) versus
potential (V). Electrochemical capacitances were calculated by averaging the
magnitudes of the currents measured at 0.1 V during the forward and reverse scans
and then dividing by both the scan rate and the electrode area.
An electrical double layer is formed at the solid-solution interface during
cyclic voltammetry. The double layer is modeled by Helmholtz theory as a parallel
plate capacitor:
14

εε0
C (2)
d
where C is the capacitance per unit area, d is the effective thickness of the separating
medium, ε is the dielectric constant of the medium, and ε0 is the vacuum
permittivity. 90 If a monolayer is not densely-packed and has defects, the effective
barrier thickness, d, will be less than expected from calculating molecular lengths of
the adsorbates because ions in the electrolyte solution can penetrate through the
defects to the electrode surface resulting in a higher measured capacitance. Less
permeation of ions through the monolayer and therefore a lower capacitance will be
measured if the monolayer is densely-packed and defect-free.
Collaborations
I have had the privilege of collaborating with several people during the course
of my doctoral research as outlined in this section.
The cyclic voltammetry measurements that were used to estimate the
electrochemical capacitances reported in Chapter 2 were performed in collaboration
with Mr. Matthew Pellow, a graduate student in Prof. Dan Stack‟s research group. I
was present and assisted during all of the cyclic voltammetry experiments. Mr. Pellow
and Mr. Eric Stenehjem, another graduate student in the Stack group, have been using
my method to vapor deposit azide-terminated monolayers onto ITO electrodes as
platforms for covalently attaching alkyne-terminated, inorganic metal ligand
complexes for electrocatalytic screening.
The work in Chapter 3 was performed in collaboration with Ms. Sujatha Raghu,
a local middle school teacher who enthusiastically performed research in the Chidsey
15

group over two summers in 2006 and 2008. Ms. Raghu was involved with the first
experiments that I performed on azide-terminated monolayers as her first day in the
Chidsey group was also my first day. Ms. Raghu was particularly involved with the
ellipsometry and water contact angle measurements.
This thesis is the result of a collaboration that was established between Dr. Tim
Harris then at Helicos BioSciences and Prof. Chidsey at Stanford before I joined the
Chidsey group. As a result of this collaboration with Helicos, I have had the privilege
of working with two outstanding collaborators, Dr. Erin Artin and Dr. Tim Harris. Dr.
Harris acted as a co-advisor for me during the collaboration and provided technical
guidance and support in parallel to my principal advisor, Prof. Chidsey. Dr. Artin
synthesized alkyne-terminated oligonucleotides and fluorescent dyes and shipped
them to me for experiments at Stanford. My role in the collaboration was the
development of best practices for the reproducible deposition of azide-terminated
monolayers onto glass substrates while maintaining a low fluorescent background. I
used bulk fluorescence measurements at Stanford to optimize CuAAC reaction
conditions for the covalent attachment of oligonucleotides and to develop methods to
reduce nonspecific adsorption. Technology developed at Stanford was transferred to
Helicos through onsite visits and correspondence. Dr. Harris and Dr. Artin performed
more sensitive single molecule fluorescence measurements on glass surfaces using
instruments at Helicos.
Mr. David Lapham, an undergraduate student in the Chidsey group, showed
that the covalently attached oligonucleotides were viable for hybridization with
complementary oligonucleotides as measured using bulk fluorescence spectroscopy.
16

Furthermore, Dr. Artin performed single molecule sequencing runs at Helicos on glass
surfaces modified with azide-terminated monolayers. Preliminary experiments
suggested that the azide-terminated monolayers exhibited higher resistance to the
nonspecific adsorption of dye-labeled nucleotides after reaction with propargyl alcohol
than the commercially-produced, epoxy-terminated monolayers that Helicos uses as
their standard substrate. Unfortunately, this collaboration came to an abrupt and
untimely end in December 2008. Chapter 4 discusses some of the work performed
during the collaboration with Helicos.
Mr. Minsub Chung, a graduate student in Prof. Steve Boxer‟s group in the
Chemistry department at Stanford, established an interesting collaboration with me. I
attached alkyne-terminated oligonucleotides and ethynyl phosphonic acid to azide-
terminated monolayers on glass surfaces. Mr. Chung used these surfaces as supports
for tethered lipid bilayers by rupturing vesicles using hybridization between
complementary oligonucleotides displayed on the surface and on the vesicles. This
work is not discussed in this thesis but resulted in a publication on which I am second
author.
I also collaborated with a Chidsey group student, Mr. Jonathan Prange, on the
solution deposition of azide-terminated phosphonic acid monolayers onto ITO
electrode surfaces. I synthesized p-azidophenylphosphonic acid and performed initial
experiments with it. Mr. Prange followed up on this work upon joining the Chidsey
group, which is discussed in detail in his thesis.
17

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25

Chapter 2: Deposition of Dense Siloxane Monolayers from Water and
Trimethoxyorganosilane Vapor*
Preface
This chapter of my dissertation is from a manuscript that was recently accepted
to the scientific journal Langmuir. I am the first author of this publication and am
responsible for most of the writing and preparation. The other authors are Matthew A.
Pellow, Professor T. Daniel P. Stack, and Professor Christopher E. D. Chidsey. I was
involved in all of the experiments that were conducted for this manuscript. My main
contributions to this manuscript include: pursuing vapor deposition, synthesis of
tetradecyltri(deuteromethoxy)silane, using MgSO4·7H2O as a controlled water source
and studying the effect of its addition, and designing the sample holder for Brewster‟s
angle transmission FTIR spectroscopy.
Prof. Chidsey proposed using cyclic voltammetry to estimate electrochemical
capacitance as a measure of monolayer densification on indium tin oxide (ITO)
electrodes. Matthew A. Pellow performed the cyclic voltammetry experiments, but I
was present and assisted with all experiments. Prof. Chidsey and I developed the idea
of using the selectively deuterated silane for studying the hydrolysis of
trimethoxysilanes. Prof. Chidsey also provided significant guidance in interpreting and
planning experiments as well as in the writing and preparation of the manuscript.
* Reproduced with permission from Lowe, R.D.; Pellow, M.A.; Stack, T.D.P.;
Chidsey, C.E.D. “Deposition of Dense Siloxane Monolayers from Water and
Trimethoxyorganosilane Vapor,” Langmuir, 2011, 27(16), 9928-9935. ©Copyright
2011 American Chemical Society.
26

Abstract
A convenient, laboratory-scale method for the vapor deposition of dense
siloxane monolayers onto oxide substrates was demonstrated. This method was
studied and optimized at 110°C under reduced pressure with the vapor of
tetradecyltri(deuteromethoxy)silane, (CD3O)3-Si-(CH2)13-CH3, and water from the
dehydration of MgSO4·7H2O. Ellipsometric thicknesses, water contact angles, Fourier
transform infrared (FTIR) spectroscopy, and electrochemical capacitance
measurements were used to probe monolayer densification. The CD3 stretching mode
in the FTIR spectrum was monitored as a function of the deposition time and amounts
of silane and water reactants. This method probed the unhydrolyzed methoxy groups
on adsorbed silanes. Excess silane and water were necessary to achieve dense,
completely hydrolyzed monolayers. In the presence of sufficient silane, an excess of
water above the calculated stoichiometric amount was necessary to hydrolyze all
methoxy groups and achieve dense monolayers. The excess water was partially
attributed to the reversibility of the hydrolysis of the methoxy groups.
27

Introduction
We have developed a convenient procedure for vapor-depositing dense siloxane
monolayers onto oxide surfaces and a Fourier transform infrared (FTIR) spectroscopic
probe for monitoring the hydrolysis of methoxy groups during deposition. Siloxane
monolayers have been used in several applications ranging from chromatography 1 to
the surface attachment of bioanalytical probes. 2,3 These monolayers control the
properties of oxide surfaces by introducing organic functionality. 4-6 This is
accomplished using silane monomers with headgroups that bond to the surface and
crosslink among themselves and tailgroups that present desired chemical moieties. The
ability to investigate and control the underlying interfacial chemistry allows the
reproducible deposition of siloxane monolayers of controlled density suitable for
specific applications.
Siloxane monolayer formation is a multistep process beginning with organosilane
monomers possessing one to three hydrolyzable groups, such as chloro or alkoxy
groups. 7 These hydrolyzable groups react directly with surface hydroxyl groups to
form siloxane or other silicon to metal oxide linkages. The hydrolyzable groups also
react with water to produce silanols on the organosilanes. 8 These silanols can undergo
condensation reactions with surface hydroxyl groups or with silanols on neighboring
organosilanes to form additional siloxane linkages and water as a byproduct.
Monolayers formed from trifunctional silanes, such as the ones used in this study,
have been described as two-dimensional, network polymers with occasional bonds to
the oxide surface. 9
28

Because reaction of the hydrolyzable groups should be necessary to form surface-
attached, two-dimensional network polymers, the amount of water is known to be an
important variable for the reproducible deposition of siloxane monolayers from
trifunctional silane monomers. With insufficient water available, unhydrolyzed alkoxy
groups on the silanes could sterically hinder dense monolayer formation. This would
be similar to the case of monofunctional alkyldimethylsilanes where the bulky
dimethylsilyl groups prevented tight alkane chain packing. 10
Numerous studies have discussed the importance of water in depositing siloxane
monolayers from solutions. 4,11,12 When sufficient water was present, larger
ellipsometric thicknesses 10,13 and contact angles 14 were measured and indicated the
deposition of denser monolayers compared to more anhydrous cases. However, those
measurements did not provide detailed information about the chemical bonds at the
interface. X-ray photoelectron spectroscopy (XPS) 10,14-16 and FTIR spectroscopy 17,18
have been used to confirm the hydrolysis of Si-Cl bonds during solution deposition of
trichlorosilanes. Using FTIR spectroscopy, Pasternack et al. found unhydrolyzed
ethoxy groups after solution deposition of triethoxysilanes under anhydrous
conditions. 19 However, the FTIR peaks assigned to residual C-H stretching modes of
the ethoxy groups were obscured by peaks associated with Si-O-Si and other C-H
stretching modes. In this paper, we describe a clearer method of monitoring the
hydrolysis of alkoxy groups. Deuteration of hydrolyzable methoxy groups gives a C-D
stretching mode that is shifted into an unobscured region of FTIR spectra.
Siloxane monolayers have been deposited from dilute solutions 20 and from the vapor
phase. 21 Monolayer deposition from the vapor phase is attractive because there is less
29

siloxane oligomer particle contamination than during solution-phase deposition. 22-24
As in solution-phase deposition, the amount of water is an important variable for
vapor-phase siloxane monolayer deposition. Vapor-phase deposition with 22,25-33 and
without 16,34-42 the deliberate addition of water vapor has been reported, but the effect
of water addition has not been studied as thoroughly as in the solution case.
In this paper, we present a simple method for vapor-depositing siloxane monolayers
with controlled amounts of water vapor and show that dense siloxane monolayers are
not deposited unless sufficient water is present to hydrolyze the methoxy groups and
sufficient silane is present to densify the monolayers. The hydrolysis of
tetradecyltri(deuteromethoxy)silane, (CD3O)3-Si-(CH2)13-CH3, is monitored after
vapor-phase deposition using FTIR spectroscopy on silicon oxide surfaces.
Specifically, we study how the deposition duration and the initial amounts of water
and silane reactants effect methoxy group hydrolysis and monolayer completion.
Ellipsometric thicknesses, FTIR spectroscopy, water contact angles, and
electrochemical capacitance measurements are used to probe monolayer densification.
Experimental
Silane Synthesis
Tetradecyltri(deuteromethoxy)silane was prepared from n-tetradecyltrichlorosilane
(Gelest) via methanolysis of the trichloro groups following published procedures for
making similar alkoxysilanes. 43,44 2.00 g of tetradecyltrichlorosilane (6 mmol) in 15
mL of dry pentane was slowly added to a solution of 0.67 g (19 mmol) deuterated
methanol, CD3OD (99.6+ atom % D, Acros), and 1.85 g (19 mmol) of dry pyridine in
75 mL of dry pentane while stirring under nitrogen and cooled in an ice water bath.
30

The solution was allowed to stir overnight at room temperature under a nitrogen
atmosphere, filtered to remove pyridinium chloride, and then concentrated by rotary
evaporation. The crude product was purified by two successive vacuum distillations.
See the Supporting Information for nuclear magnetic resonance (NMR) and FTIR
characterization.
Surface Cleaning
Double and single-side polished Si(100) wafers were used as received or after prior
deposition experiments and cleaned in an oxygen plasma (GaLa Instrumente Plasma
Prep5) for 10 min prior to deposition using 50 percent relative power with a dioxygen
flow rate of 100 std. mL/min at a pressure of 0.25 mbar. These surfaces were exposed
to atmospheric air for approximately 15 min after cleaning in order to determine
effective refractive indices of the substrates with an ellipsometer. This cleaning
method resulted in hydrophilic surfaces with water contact angles approaching 0°.
Indium tin oxide (ITO) films supported on glass substrates had sheet resistances of 8-
12 ohms (Delta Technologies). The ITO surfaces were plasma cleaned with the same
procedure as the silicon oxide surfaces.
Monolayer Vapor Deposition
Siloxane monolayers were vapor-deposited onto silicon oxide and ITO surfaces in
o-ring sealed, glass vacuum desiccators fitted with Teflon stopcocks (Jencons part
number 250-048) with internal volumes of about 600 mL (Figure 1). These chambers
were oven dried for a minimum of 4 h at 140°C in air and cooled for no more than 10
min in air before use. Neat silane (100 µL) was pipetted onto and absorbed into 42.5
mm diameter Whatman filter paper in the bottom of the desiccator. Various masses of
31

MgSO4·7H2O (Fisher biochemical grade) were placed in a foil boat in the bottom of
the desiccator as a water source for the hydrolysis reaction. Plasma-cleaned surfaces
were placed on a metal rack supported above the silane liquid and hydrated salt in the
glass vacuum chamber. The desiccator was then evacuated through a rubber hose on a
glass vacuum line with a liquid nitrogen-trapped mechanical pump for approximately
60 seconds. The final pressure at the trap was 1 Torr. The Teflon valve on the
desiccator was closed, and the chamber was placed in a 110°C preheated oven (Forma
Scientific) for various periods of time from 1 to 24 h. After deposition, the valve was
opened to ambient air to return the chamber to atmospheric pressure and remove the
samples. Ashurst et al. used a related but more elaborate setup. 25 Their chamber was
modified with water and silane reactant inputs from external sources similar to other
systems that have been used to introduce water during vapor depositions.
Deposition Chamber
Oxide Surfaces
RSi(OCD 3 ) 3 (l) MgSO 4 ·7H 2 O(s)
32
H 2 O(g)
RSi(OCD 3 ) 3 (g)
- CD 3 OH(g)
Figure 1. Schematic of the glass vacuum chamber that was used for siloxane
monolayer vapor depositions onto oxide surfaces from liquid silane and MgSO4·7H2O
reactants. An incomplete monolayer with unhydrolyzed methoxy groups is shown in
the schematic in the middle. A dense, completely hydrolyzed monolayer (drawing

adapted from Ulman 8 ) deposited in the presence of sufficient silane and MgSO4·7H2O
is shown on the right.
Treatment of Monolayers with Methanol Vapor
Substrates onto which monolayers had been previously vapor deposited were placed
in a clean, dry desiccator chamber. The glass desiccator was evacuated to 1 Torr, and
the Teflon valve was closed as in the monolayer deposition experiments. A rubber
septum was then placed on the Teflon valve. The septum was then punctured with a
needle on a syringe containing pure deuterated methanol or a premixed deuterated
methanol-water liquid solution. The Teflon valve on the vacuum desiccator was then
opened, which evacuated the contents of the syringe into the desiccator under vacuum.
The Teflon valve was then closed, and the rubber septum and syringe were removed
from the top of the desiccator. The desiccator was then placed into a preheated 110°C
oven for 12 h.
Surface Characterization
FTIR spectra were obtained with a Bruker Vertex 70 spectrometer using a KBr
beam splitter and a deuterated triglycine sulfate (DTGS) detector. Spectra were
collected in transmission mode through the silicon with the samples at the silicon
Brewster‟s angle (74°) 45 using p-polarized light from a wire-grid polarizer (Specac
KRS-5). For each spectrum, 1024 scans were collected at 4 cm -1 resolution.
Background spectra were collected from freshly oxygen plasma-cleaned, double side
polished Si(100) crystals in the same orientation.
Ellipsometry measurements were performed using a Gaertner L116 ellipsometer at
70° angle of incidence. Effective real and imaginary refractive indices of the Si(100)
33

substrates were determined immediately after plasma cleaning but before siloxane
monolayer vapor deposition. Measurements after vapor deposition, rinsing with
toluene and isopropyl alcohol, and drying in a stream of nitrogen were used to
determine monolayer thicknesses. A film refractive index of 1.46 was used for all
thickness calculations. At least three measurements were performed on each sample to
obtain average substrate refractive indices or sample thicknesses.
Advancing sessile drop water contact angles were measured with a Ramé-Hart
model 100 goniometer using water from a four-bowl Millipore purification system.
Water droplets were dispensed from a syringe with a flat-tipped needle. Fresh water
was obtained from the Millipore system for each measurement session. At least two
measurements were made on each sample on both sides of the droplets and averaged.
Cyclic voltammetry experiments were conducted using either a Bioanalytical
Systems model CV-50W or a Pine Instrument Company model AFCBP1 potentiostat.
ITO surfaces with an exposed area of 0.21 cm 2 were used as the working electrodes in
a three-electrode electrochemical cell. Platinum wire was used as the counter electrode,
and all potentials measured were with respect to a Ag/AgCl/saturated KCl reference
electrode. Cyclic voltammograms were collected over a scan range of -0.1 V to 0.4 V
versus the reference electrode (Figure S3 in the Supporting Information). The reported
electrochemical capacitances were calculated by averaging the magnitudes of the
currents measured at 0.1 V during the forward and reverse scans and then dividing by
both the scan rate and the electrode area. Measurements were performed under
ambient laboratory conditions using an electrolyte solution of 0.1 M NaClO4 in water.
34

Results
Time Dependence of Ellipsometric Thickness, C-D Stretch and Water Contact
Angle
Figure 2a is a plot of ellipsometric thickness versus deposition time for monolayers
vapor-deposited from 100 µL tetradecyltri(deuteromethoxy)silane, without and with
0.5 g MgSO4·7H2O present. At each time point, the ellipsometric thicknesses are
greater with the hydrated salt than without. A limiting thickness is reached at 12 h in
both cases, but at different values; 14.1 Å without MgSO4·7H2O and 18.2 Å with
MgSO4·7H2O.
Figure 3 shows that a peak is present at 2073 cm -1 in the FTIR spectrum after a 12 h
deposition with 100 µL silane but without MgSO4·7H2O. This peak is assigned to the
symmetric CD3 stretching mode of the deuteromethoxy groups 46,47 and is used to
monitor methoxy group hydrolysis. With 0.5 g MgSO4·7H2O present during
deposition, the CD3 absorbance is no longer observed.
Figure 2b shows the integrated absorbance of the CD3 mode at various deposition
times. The CD3 absorbance is observed at all time points without MgSO4·7H2O
present, although it decreases with increasing deposition time until reaching a limiting
value at about 4 h. In contrast, the CD3 absorbance is not observed at any time point
with 0.5 g MgSO4·7H2O.
Figure 2c is a plot of the water contact angle versus deposition time. Before
deposition, the freshly plasma-cleaned silicon oxide surfaces exhibit water contact
angles approaching zero. After deposition, water contact angles are greater than 100°
in all cases after 1 h, but are lower for monolayers vapor-deposited without
35

MgSO4·7H2O than for those deposited with MgSO4·7H2O. The contact angles plateau
after 12 h at 104° without MgSO4·7H2O and 109° with MgSO4·7H2O.
Integrated CD 3 Absorbance / cm -1
20
16
12
8
4
0
1.6x10 -3
1.2x10 -3
8.0x10 -4
4.0x10 -4
Water Contact Angle / °
Ellipsometric Thickness / Å
0
120
100
80
60
40
20
0
(a)
(b)
(c)
without MgSO 4 ·7H 2 O
with 0.5 g MgSO 4 ·7H 2 O
0 5 10 15 20 25
36
Time / h
Figure 2. Time dependence of 100 µL (CD3O)3-Si-(CH2)13-CH3 monolayer vapor
deposition onto silicon oxide without and with 0.5 g MgSO4·7H2O present monitored

using (a) ellipsometric thickness, (b) integrated area of the CD3 FTIR symmetric
stretch, and (c) water contact angle. The lines are guides to the eye. Error bars
represent one standard deviation.
C-H Stretch
Figure 3 also shows the C-H stretching region for siloxane monolayers formed by
12 h vapor depositions. The intensities of the peaks in the 2800-3000 cm -1 range are
lower for depositions performed without MgSO4·7H2O than for those with
MgSO4·7H2O. In addition, the peak frequencies for the symmetric and asymmetric
methylene stretches shift down 3 cm -1 from 2854 and 2924 cm -1 without MgSO4·7H2O
to 2851 and 2921 cm -1 with 0.5 g MgSO4·7H2O.
Absorbance
5.0x10 -5
0
without MgSO 4 ·7H 2 O
with 0.5 g MgSO 4 ·7H 2 O
2000 2050 2100 2150 2800 2850 2900 2950 3000
Frequency / cm -1
37
Frequency / cm -1
1.0x10 -3
5.0x10 -4
Figure 3. Carbon-deuterium and carbon-hydrogen stretching regions of the p-
polarized Brewster‟s angle transmission FTIR spectra of monolayers vapor-deposited
onto silicon oxide for 12 h from 100 µL (CD3O)3-Si-(CH2)13-CH3 without and with 0.5
g MgSO4·7H2O present.
0
Absorbance

Effect of Mass of MgSO4·7H2O
The plateau of the ellipsometric thickness at long deposition time with 100 L
silane varies with the mass of MgSO4·7H2O (Figure 4a, circles). As the mass of
MgSO4·7H2O is increased, the ellipsometric thickness increases until a plateau is
reached with 0.5 g MgSO4·7H2O. Figure 4b shows the integrated absorbance of the
CD3 symmetric stretch versus the mass of MgSO4·7H2O. The mass required to
decrease the CD3 stretch to zero matches the mass required to reach the plateau in
ellipsometric thickness.
Effect of Amount and Surface Area of the Silane Source
To test whether the amount of silane limits monolayer growth, experiments were
performed with varying volumes of silane. Above 50 µL of silane, the ellipsometric
thickness is independent of the amount of silane and follows the dependence on
MgSO4·7H2O described above (Figure 4a). However, with only 10 µL silane, the
thickness is equal to the thickness observed with 100 L silane in the absence of
MgSO4·7H2O but does not increase with added MgSO4·7H2O. Reduced CD3
absorbances are observed for 50 µL and less silane with 0.05 g MgSO4·7H2O present
as shown in Figure 4b. With 0.5 g MgSO4·7H2O, the CD3 absorbance is reduced to the
level of noise for all silane volumes.
To test for any role of the surface area of the liquid silane, its exposed area was
reduced by at least a thousand fold by placing the silane into a glass capillary sealed at
one end and held with the open end upwards during deposition. Experiments using 10
µL silane with 0.05 g MgSO4·7H2O and 100 µL silane with 0.5 g MgSO4·7H2O yield
38

ellipsometric thicknesses and CD3 integrated absorbances equal to the values when the
silane is absorbed into filter paper.
Figure 4. (a) Plot of the ellipsometric thickness versus mass of MgSO4·7H2O added
for 12 h depositions onto silicon oxide with various initial volumes of (CD3O)3-Si-
(CH2)13-CH3. (b) Integrated FTIR symmetric CD3 stretching absorbance versus mass
of MgSO4·7H2O added for 12 h depositions with various initial volumes of the silane.
The lines are guides to the eye. Error bars represent one standard deviation.
39

Treatment of Monolayers with Methanol Vapor
To assess the reversibility of the hydrolysis of the methoxy groups, completely
hydrolyzed, partial monolayers deposited from 10 µL silane and 0.05 g MgSO4·7H2O
for 12 h were treated with a large excess of pure CD3OD or mixtures of CD3OD and
H2O vapor at 110°C for 12 h. 48 After treatment with pure CD3OD vapor, CD3
stretching is observed with an integrated absorbance of 3.1x10 -4 cm -1 and an
ellipsometric thickness of 14 Å. Exposure to 10:1 and 3:1 molar ratios of CD3OD:H2O
result in CD3 integrated absorbances of 2.3x10 -4 and
1.1x10 -4 cm -1 , respectively. Negligible CD3 absorbance is observed after 12 h
exposure to 1:1 CD3OD:H2O.
Electrochemical Capacitance
To assess the permeability of monolayers formed by silane vapor deposition, cyclic
voltammetry was performed on conductive ITO samples that were plasma cleaned and
vapor deposited with the silane in the same manner. Figure 5 is a plot of the
electrochemical capacitance versus scan rate determined by cyclic voltammetry.
Capacitances are highest for monolayers deposited without the addition of
MgSO4·7H2O. When monolayers are deposited in the presence of 0.5 g MgSO4·7H2O,
the measured capacitances decrease. A further decrease in the capacitances is observed
for surfaces exposed to a second 12 h deposition with 100 L of fresh silane and 0.5 g
of fresh MgSO4·7H2O. The decrease in capacitance is accompanied by an increase in
the intensity of the asymmetric methylene stretch by less than 10 percent in the FTIR
spectrum after the second deposition with no discernible changes to the peak positions
or widths of the methylene stretching modes (Figure S4 in the Supporting
40

Information). The ellipsometric thicknesses and the water contact angles are also
unchanged after the second deposition. Exposure of the surfaces to three repeated 12 h
depositions with fresh silane and fresh MgSO4·7H2O results in the same capacitance
as two repeated depositions, suggesting that a minimum limiting value of the
capacitance is reached after two repeated depositions.
Capacitance / F/cm 2
20 1x without MgSO 4 ·7H 2 O
15
10
5
0
1x with 0.5 g MgSO 4 ·7H 2 O
2x with 0.5 g MgSO 4 ·7H 2 O
3x with 0.5 g MgSO 4 ·7H 2 O
10 100 1000
Scan Rate / mV/s
Figure 5. Plot of the electrochemical capacitance versus scan rate during cyclic
voltammetry after 12 h depositions of 100 µL (CD3O)3-Si-(CH2)13-CH3 onto ITO
electrodes without and with 0.5 g MgSO4·7H2O present. The squares and triangles
represent the capacitances measured after two and three repeated 12 h depositions,
each with 100 µL of fresh silane and fresh 0.5 g MgSO4·7H2O. Error bars represent
one standard deviation.
41

Discussion
Measurements of ellipsometric thickness show that incomplete monolayers are
formed if insufficient MgSO4·7H2O is present. Dense monolayers are deposited with
100 µL silane and 0.5 g MgSO4·7H2O as inferred from the plateau of the ellipsometric
thickness at 18.2 Å independent of further increases in deposition time, amount of
silane or amount of MgSO4·7H2O. This vapor-deposited thickness is consistent with
literature values which range from 18 Å to 20 Å for monolayers solution-deposited
from tetradecyltrichlorosilane. 14,15 We infer that dense monolayers form only when
sufficient water to hydrolyze all methoxy groups is present, and sufficient silane is
present for the time required to densify the monolayer. When sufficient silane is
present, incomplete monolayers can nonetheless result from the steric crowding of
unhydrolyzed methoxy groups or from insufficient water to form additional siloxane
bonds.
FTIR spectroscopy was used to monitor the hydrolysis of methoxy groups during
deposition. From the observation of CD3 stretching in FTIR spectra measured on
monolayers prepared in the absence of sufficient MgSO4·7H2O, we infer that
unhydrolyzed methoxy groups remain on some of the adsorbed silanes. These may
prevent monolayer completion. The decrease of the CD3 integrated absorbance with
increased deposition time without MgSO4·7H2O present (Figure 2b) suggests that
methoxy groups are hydrolyzed over time by adventitious water in the chamber or on
the silicon oxide surfaces. However, adventitious water is insufficient to hydrolyze all
methoxy groups as indicated by the residual CD3 absorbance for 100 µL silane. The
deliberate addition of a water source is necessary to deposit dense monolayers.
42

Increasing the mass of MgSO4·7H2O above the approximately 0.5 g required for
complete removal of the methoxy groups on the surface yields no increase in the
ellipsometric thickness and provides further support for the possibility that unreacted
methoxy groups are responsible for incomplete monolayer deposition.
The hydrolysis of methoxy groups and completion of the monolayer are correlated
with an observed shift of methylene stretching to lower frequency. It is known that, as
the packing of alkyl chains becomes denser, the positions of the symmetric and
asymmetric methylene stretches shift to lower frequency. 49-52 In the absence of
MgSO4·7H2O, the methylene stretching frequencies are characteristic of liquid-like
packing of alkyl chains. With the addition of sufficient MgSO4·7H2O for complete
hydrolysis, the methylene stretching frequencies shift to lower frequency and are
similar to previous reports 53,54 for dense monolayers deposited from solutions of
alkyltrichlorosilanes of similar chain length. This further supports our conclusion that
incomplete monolayers are formed without sufficient MgSO4·7H2O present.
Water contact-angle measurements on monolayers deposited from 100 µL silane
with sufficient MgSO4·7H2O are equal to those measured for dense alkylsiloxane
monolayers deposited from solution 15 and provide additional evidence for improved
packing of the monolayers. Lower contact angles indicative of less dense monolayers
are measured without deliberate addition of MgSO4·7H2O.
The amount of silane required to form a dense monolayer when there is sufficient
MgSO4·7H2O is approximately 100 µL (Figure 4a), which is 2 orders of magnitude
above the calculated amount needed to coat all surfaces in the deposition chamber
with a dense monolayer of the silane. 55 The volatile silane is presumably depleted by
43

hydrolysis and condensation reactions in the reservoir of liquid silane leading to cross-
linked siloxanes with low vapor pressure. The lifetime of the silane source must be
sufficiently large to provide volatile silane for the time required for monolayer
densification. This is illustrated by experiments that use 10 µL silane with 0.05 g and
0.5 g MgSO4·7H2O. In those experiments, complete methoxy group hydrolysis is
observed, but ellipsometric thicknesses are measured that are similar to monolayers
deposited from 100 µL silane without MgSO4·7H2O. Experiments with the silane
liquid source contained in a glass capillary, rather than being absorbed into filter paper,
do not result in observable differences in the monolayers, suggesting that the surface
area of the silane source is not limiting its lifetime.
To further assess the lifetime of the silane source, silicon oxide and ITO surfaces
were subjected to two repeated 12 h vapor depositions. Fresh reactants, 100 µL silane
and 0.5 g MgSO4·7H2O, were used in each deposition. Exposure to a second
deposition results in an increase in the intensity of the asymmetric methylene stretch
of less than 10 percent with no discernible changes to the methylene stretching peak
positions or widths, the ellipsometric thicknesses, or the water contact angles of
monolayers deposited onto silicon oxide surfaces. The work of Hong et al. 26
motivated us to pursue a more sensitive measure of monolayer densification. They
reported no changes in the ellipsometric thicknesses or water contact angles measured
on vapor-deposited monolayers by increasing the deposition time beyond 12 h.
However, an increase in the blocking of atomic layer deposition was observed on
monolayers deposited for longer than 12 h.
44

Electrochemical capacitance measurements were performed to test the resistance of
vapor-deposited siloxane monolayers on ITO to ion permeation. The measured
capacitances decrease with increasing scan rate (Figure 5). We attribute this decrease
to a decrease in the time for permeation with higher scan rates. 56 Electrochemical
capacitances are highest for monolayers deposited without MgSO4·7H2O, from which
we infer that these monolayers are most permeable to ions in solution. This
observation agrees with the ellipsometric, FTIR spectroscopic, and water contact angle
measurements indicating incomplete monolayers. The denser monolayers formed with
0.5 g MgSO4·7H2O are more resistant to ion permeation. Exposing monolayers
formed with 0.5 g MgSO4·7H2O to a second 12 h deposition with fresh 100 µL silane
and fresh 0.5 g MgSO4·7H2O results in even greater resistance to ion permeation. We
infer that the electrochemical capacitance is a more sensitive probe for monolayer
densification than the other measurements used because a difference in the
electrochemical capacitance is clearly observed after the second deposition while only
a subtle increase in the asymmetric methylene stretching intensity in the FTIR
spectrum is observed with no discernible changes to the methylene stretching peak
positions or widths, ellipsometric thicknesses, or water contact angles.
The lowest capacitance measured, 2.0 µF·cm -2 , is higher than the value of 1.2
µF·cm -2 , for thiol monolayers of the same chain length on gold. 51 We infer that vapor-
deposited siloxane monolayers on ITO contain more pinhole and gauche defects that
allow ion permeation than their thiol counterparts on gold. Previous reports of
capacitance values measured on siloxane monolayers solution-deposited onto
electrode surfaces vary widely 57-59 from 1.1 to 24 µF·cm -2 , highlighting the sensitivity
45

of the monolayer packing in siloxane monolayers to the details of the deposition
method.
We also find that an excess of water above the calculated stoichiometric minimum
amount of water is required to achieve complete hydrolysis and dense monolayers. For
example, the complete hydrolysis of 100 µL silane (0.27 mmol) should
stoichiometrically require 1.5 times as much water (0.40 mmol). 60 However, we
observe in Figures 4a and 4b that dense monolayer formation requires approximately
0.5 g MgSO4·7H2O which, upon partial dehydration at 110°C, 61-63 yields 9 mmol of
water, or about twenty times as much water as required for complete hydrolysis of the
silane.
What determines the amount of water required to form dense monolayers when
sufficient silane is present? Equilibrium effects might explain the excess of water
required to achieve complete hydrolysis. Methanol generated as a byproduct of
hydrolysis reactions accumulates in the deposition chamber over time and could drive
the reverse methoxylation reaction. We have confirmed the reversibility of the
hydrolysis of methoxy groups by exposing completely hydrolyzed, partial monolayers
to pure CD3OD and CD3OD/H2O vapor mixtures and observing the CD3 stretch in
FTIR spectra. We attribute the reappearance to methoxylation of free silanols on the
surface. The CD3 stretch is observed after exposure to CD3OD:H2O vapor mixtures
with molar ratios of 3:1 and higher but not with a 1:1 ratio. From these data, we
estimate that the equilibrium for methoxy group hydrolysis is mildly favorable with an
equilibrium constant of approximately 4. 64 This is in agreement with determinations of
46

the equilibrium constant for alkoxy group hydrolysis in solution on the order of 10. 65-
70
While the equilibrium argument predicts the qualitative trend of increased density
with increased water, the equilibrium constant we calculate from reversing the
hydrolysis does not quantitatively explain the data. For instance, with 50 µL silane
and 0.05 g MgSO4·7H2O, the methanol to water ratio is expected to be 0.6, well below
the level required to inhibit the hydrolysis reaction (Table S1 in the Supporting
Information). One possibility is that water release from the MgSO4·7H2O is rate
limiting. In that case, the liquid silane may remove most of the water from the vapor
phase with the result that the silanes in the monolayer remain methoxylated until the
liquid silane is exhausted and monolayer growth terminates. Increasing the mass of
MgSO4·7H2O and the amount of water released per unit time might maintain a greater
fraction of free silanols in the monolayer at early time and promote monolayer growth.
Conclusions
In summary, we showed that the addition of water was necessary during siloxane
monolayer vapor deposition to hydrolyze methoxy groups and form dense monolayers.
Water was deliberately introduced to vapor depositions through the dehydration of
MgSO4·7H2O. We developed a probe to measure the hydrolysis of deuterium-labeled
methoxysilanes. This allowed the monitoring of CD3 stretching by FTIR spectroscopy
as a function of time, mass of MgSO4·7H2O, and amount of silane. With sufficient
silane present, ellipsometric thicknesses consistent with dense monolayers were
measured when a ten-to-twenty fold excess of water above the stoichiometric amount
was present. Lower frequency methylene stretching modes, higher water contact
47

angles, and lower electrochemical capacitances indicating denser monolayers were
achieved when enough silane and water were present. Electrochemical capacitance
measurements were the most sensitive to changes in the density of the siloxane
monolayers. The excess water required was at least in part due to the reversibility of
the hydrolysis of the methoxy groups.
We provide a simple laboratory method to deposit dense siloxane monolayers using
optimized amounts of silane and water and repeated deposition cycles if needed. We
are currently using this method to deposit mixed, azide-terminated monolayers onto
oxide surfaces, which can be further functionalized with a broad array of groups by the
Cu(I)-catalyzed azide-alkyne cycloaddition reaction.
Acknowledgment. This work was supported by funding from Helicos BioSciences
Corporation. The authors acknowledge NSF grant CHE-0639053 for support of FTIR
equipment. The authors also acknowledge NSF grant DMR-0213618 to the Center on
Polymer Interfaces and Macromolecular Assemblies for use of the plasma cleaner and
ellipsometer. Useful discussions with Dr. Erin Artin and Dr. Tim Harris are
acknowledged.
Supporting Information Available: NMR and FTIR spectra of tetradecyl-
tri(deuteromethoxy)silane. Cyclic voltammogram and FTIR spectrum of a siloxane
monolayer after two repeated 12 h vapor depositions. Time dependence of
MgSO4·7H2O dehydration. Table of expected CH3OH:H2O ratios in the deposition
48

chamber based on stoichiometry. This material is available free of charge via the
Internet at http://pubs.acs.org/.
Supporting Information
Figure S1. Attenuated total reflectance (ATR) FTIR spectrum of the bulk, liquid
tetradecyl-tri(deuteromethoxy)silane used for siloxane monolayer vapor depositions.
49

Figure S2. NMR spectrum of tetradecyl-tri(deuteromethoxy)silane that was
synthesized and used for siloxane monolayer vapor depositions. The spectrum was
obtained on a 300 MHz Varian Inova spectrometer in CDCl3 solvent.
50

Current Density / A·cm -2
10
5
0
-5
-10
-0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5
Potential / V vs. Ag/AgCl/saturated KCl
Figure S3. Plot of the current versus applied potential measured during cyclic
voltammetry for a siloxane monolayer on ITO exposed to 0.1 M NaClO4 in water at a
scan rate of 1000 mV/s. The siloxane monolayer was formed from two repeated 12 h
vapor depositions, each with 100 µL of fresh (CD3O)3-Si-(CH2)13-CH3 and fresh 0.5 g
MgSO4·7H2O.
51

Absorbance
1.0x10 -3
5.0x10 -4
0
2x with MgSO 4 ·7H 2 O
1x with MgSO 4 ·7H 2 O
2800 2850 2900 2950 3000
Frequency / cm -1
Figure S4. Carbon-hydrogen stretching region of the p-polarized Brewster‟s angle
transmission FTIR spectra of siloxane monolayers on silicon oxide after one 12 h
vapor deposition (solid line) and two repeated 12 h vapor depositions (dotted line),
each with 100 µL of fresh (CD3O)3-Si-(CH2)13-CH3 and fresh 0.5 g MgSO4·7H2O.
52

equiv H 2 O : equiv MgSO 4 ·7H 2 O
6
5
4
3
2
1
0
0 2 4
Time / h
Figure S5. Time dependence of the dehydration of MgSO4·7H2O at 110°C under
reduced pressure starting with 0.5 g MgSO4·7H2O. The dehydration of the salt was
monitored gravimetrically. The plateau at about 4.5 equivalents of water released per
equivalent of initial MgSO4·7H2O was used for all stoichiometry calculations. The
line is a guide to the eye.
53

Table S1. Calculated CH3OH:H2O Ratio in Deposition Chamber Assuming Complete
Hydrolysis and Condensation
volume silane
mass MgSO4·7H2O
10 µL
50 µL
0 g 0.005 g 0.050 g 0.25 g 0.50 g 1.0 g
0.092
0.56
54
0.0088
0.045
100 µL ∞ ∞ 1.6 0.19 0.092 0.045
300 µL
∞
Table S1 shows the calculated CH3OH:H2O ratio expected at long deposition time in
the silane deposition chamber assuming a water:trimethoxysilane stoichiometric ratio
of 1.5:1 for conditions used in this study. Entries in the table with „∞‟ indicate pure
methanol should remain because there was enough silane present to consume all of the
water in the system. Therefore, it is not surprising that CD3 stretching was observed
after those experiments with less than stoichiometric water present. However, CD3
stretching was still observed from experiments with water in stoichiometric excess,
100 µL silane and 0.05 g MgSO4·7H2O, for example. A CH3OH:H2O ratio of 1.6 in
the deposition chamber is predicted under those conditions. With the estimated
equilibrium constant of 4.3 for the hydrolysis reaction, a SiOCD3:SiOH ratio of ~ 0.36
is predicted. Decreasing the amount of silane to 50 µL with the same amount of water
from 0.05 g MgSO4·7H2O still resulted in CD3 stretching. Under these conditions, a
SiOCD3:SiOH ratio of ~ 0.13 is predicted. Although the amount water present under
these conditions was greater than stoichiometric requirements, equilibrium predicts
that a fraction of available silanols would be methoxylated as confirmed by the
observation of CD3 stretching. No CD3 stretching was observed after lowering the
0.3

amount of initial silane to 10 µL with 0.05 g MgSO4·7H2O. Less driving force for
reversing the hydrolysis would be expected with a calculated CH3OH:H2O ratio of
0.092 and a SiOCD3:SiOH ratio of 0.021. This same ratio is expected for experiments
using 100 µL silane with 0.5 g MgSO4·7H2O which resulted in dense, completely
hydrolyzed monolayers. Dense, completely hydrolyzed monolayers were also obtained
from experiments using 300 µL silane with 0.5 g MgSO4·7H2O with higher calculated
CH3OH:H2O and SiOCD3:SiOH ratios of 0.3 and ~ 0.07, respectively.
55

References and Notes
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(48) An excess of vapor was used during these post-treatments. At 110°C, a total
pressure of about 480 Torr is expected in the deposition chamber. The total
number of moles of vapor was kept constant at 0.012 mol for all mixtures used.
The following masses of CD3OD and H2O were used in these experiments:
0.440 g CD3OD and 0 g H2O (pure CD3OD), 0.400 g CD3OD and 0.020 g H2O
(10:1 molar ratio), 0.330 g CD3OD and 0.055 g H2O (3:1 molar ratio), and
0.216 g CD3OD and 0.108 g H2O (1:1 molar ratio).
(49) Snyder, R. G.; Strauss, H. L.; Elllger, C. A. J. Phys. Chem. 1982, 86, 5145-
5150.
59

(50) Snyder, R. G.; Maroncelli, M.; Strauss, H. L.; Hallmark, V. M. J. Phys. Chem.
1986, 90, 5623-5630.
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(54) Tillman, N.; Ulman, A.; Schildkraut, J. S.; Penner, T. L. J. Am. Chem. Soc.
1988, 110, 6136-6144.
(55) A dense siloxane monolayer should exhibit an approximate coverage of 5x10 14
molecules per cm 2 based on each alkylsilane occupying an area of about 20
Å 2 . 15 Therefore, 100 µL (0.27 mmol) of the silane should be enough to cover
3x10 5 cm 2 with a monolayer. The upper limit of surface area inside the silane
vapor deposition chamber is estimated to be approximately 10 3 cm 2 .
(56) Chidsey, C. E. D.; Loiacono, D. N. Langmuir 1990, 6, 682-691.
(57) Hillebrandt, H.; Tanaka, M. J. Phys. Chem. B 2001, 105, 4270-4276.
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(59) Chen, C.; Hutchison, J. E.; Postlethwaite, T. A.; Richardson, J. N.; Murray, R.
W. Langmuir 1994, 10, 3332-3337.
(60) The calculation of 0.4 mmol water to hydrolyze 0.27 mmol (100 µL) of the
silane assumes a water:trimethoxysilane stoichiometric ratio of 1.5:1, which is
derived from the overall reaction of complete trimethoxysilane hydrolysis
(requiring water) followed by complete condensation (releasing water) to form
a polysiloxane network. The expected upper limit for the
60

water:trimethoxysilane stoichiometry is 3:1 from complete hydrolysis of the
methoxy groups and no water-producing condensation to form siloxanes. In
this case, 0.8 mmol water would be needed to hydrolyze the silane.
(61) Experiments were conducted to determine the amount of water released from
the dehydration of MgSO4·7H2O, at 110°C. Specifically, 0.5 g MgSO4·7H2O
was placed into the same deposition chamber and 110°C oven used in the
vapor deposition experiments for various periods of time. The measured mass
difference was used to determine how much water was liberated (Figure S5 in
the supporting information). Our experimental determination of the hydrated
salt releasing about 4.5 waters per formula unit agrees well with previous
reports. 62,63 In addition, loss of vacuum in the chamber was observed after the
dehydration of masses of MgSO4·7H2O slightly greater than 1.0 g as expected
from the measured dehydration stoichiometry.
(62) Paulik, J. J.; Paulik, F.; Arnold, M. Thermochim. Acta 1981, 50, 105.
(63) Emons, H.; Ziegenbalg, G.; Naumann, R.; Paulik, F. J. Therm. Anal. 1990, 36,
1265.
(64) The equilibrium constant for hydrolysis was estimated by interpolation of the
measured CD3 absorbances after exposure to CD3OD/H2O mixtures. An
interpolation was performed between the 10:1 and 3:1 CD3OD:H2O data points
to estimate the ratio where an integrated CD3 absorbance of 1.55x10 -4 cm -1 ,
one-half the absorbance after exposure to pure CD3OD, should be measured.
The result of this interpolation was a CD3OD:H2O molar ratio of 4.3:1, which
is the equilibrium constant for the reaction: RSiOCD3 + H2O ↔ RSiOH +
61

CD3OH. It is assumed that all available free silanols that would participate in
the equilibrium reaction would be methoxylated after a 12 h treatment with
excess pure CD3OD.
(65) Ro, J. C.; Chung, I. J. J. Non-Cryst. Solids 1989, 110, 26-32.
(66) Rankin, S. E.; McCormick, A. V. Macromolecules 2000, 33, 7743-7750.
(67) van Beek, J. J.; Seykens, D.; Jansen, J. B. H. J. Non-Cryst. Solids 1992, 146,
111-120.
(68) Rankin, S. E.; Sefcík, J.; McCormick, A. V. Ind. Eng. Chem. Res. 1999, 38,
3191-3198.
(69) Sanchez, J.; McCormick, A. J. Phys. Chem. 1992, 96, 8973-8979.
(70) Sefcík, J.; Rankin, S. E.; Kirchner, S. J.; McCormick, A. V. J. Non-Cryst.
Solids 1999, 258, 187-197.
62

Chapter 3: Vapor Deposition of Mixed, Azide-Terminated Siloxane Monolayers: A
Modular Strategy for Modifying Oxide Surfaces
Preface
This chapter of my dissertation is from a manuscript that will be submitted to
the scientific journal Langmuir. I am the first author of this publication and am
responsible for most of the writing and preparation. The other authors are Sujatha
Raghu, and Professor Christopher E. D. Chidsey. I was involved in all of the
experiments that were conducted for this manuscript. My main contributions to this
manuscript include: synthesis of the 11-azidoundecyltrimethoxysilane and
tetradecyltri(deuteromethoxy)silane adsorbates, optimization of the vapor deposition
conditions, demonstrating the importance of water addition, determining mixed
monolayer compositions, and optimization of copper-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction conditions.
Sujatha Raghu‟s contributions were in performing ellipsometry and water
contact angle measurements and in assisting with the first mixed monolayer vapor
deposition experiments. Prof. Chidsey proposed the idea of studying azide-terminated
siloxane monolayers on silicon oxide as part of the collaboration with Helicos
BioSciences. He also provided significant guidance in interpreting and planning
experiments as well as in the writing and preparation of the manuscript.
63

Abstract
Pure and mixed azide-terminated monolayers are deposited from water vapor
and the vapor of pure and mixed trimethoxyorganosilanes. Pure azide-terminated
monolayers are deposited from 11-azidoundecyltrimethoxysilane vapor. Denser
monolayers are achieved when MgSO4·7H2O is present as a deliberate water source
than without it, as inferred from ellipsometry, Fourier transform infrared (FTIR)
spectroscopy, and x-ray photoelectron spectroscopy (XPS) measurements. Mixed
monolayers are vapor deposited from 11-azidoundecyltrimethoxysilane and an
alkyltrimethoxysilane diluent. The relative surface compositions of the mixed
monolayers are enriched with the silane reactant with the higher vapor pressure as
probed using FTIR spectroscopy, XPS, and water contact angle goniometry. Uniform
mixing in the mixed monolayers is inferred from the quantitative Cu-catalyzed azide-
alkyne cycloaddition (CuAAC) reactions of the azide groups with 4-ethynyl-α,α,α-
trifluorotoluene with azide coverages up to approximately 40% of a monolayer.
Quantitative reactions are not observed on dense monolayers of only azide groups,
presumably due to steric hindrance.
64

Introduction
We have developed a modular method for the reproducible, covalent
attachment of a species of interest to oxide surfaces with tunable coverage. Our
method involves the vapor deposition of mixed, azide-terminated siloxane monolayers
onto oxide surfaces followed by the Cu-catalyzed azide-alkyne cycloaddition
(CuAAC) reaction to attach alkyne-terminated species of interest as shown in Figure 1.
The surface properties of oxide surfaces are often tailored with chemisorbed
monolayers presenting desired functionality at the surface. 1,2,3 These monolayers can
be formed from adsorbates with silane 4 and less frequently with phosphonic acid 5 or
carboxylic acid 6 headgroups that can form durable bonds to many metal oxide surfaces.
One approach for covalently modifying oxide surfaces is the synthesis and
deposition of unique adsorbates each time one wants to change the species or
functionality presented at the surface. However, simply tethering an adsorbate group,
such as a silane, to a species of interest does not guarantee its successful anchoring. A
more modular strategy is the formation of a monolayer presenting a specific functional
group onto which a variety of species can be covalently attached using a predictable
coupling chemistry. 7 This latter approach is advantageous because it avoids extensive
chemical syntheses in attaching a compatible adsorbate group to each species, and it
allows independent optimization of each step.
We chose the CuAAC reaction for our coupling chemistry over the multitude
of other interfacial chemistries that have been used to covalently attach species to
monolayers. 8 The CuAAC reaction is the most well-known „click‟ reaction and was
discovered independently by Sharpless 9 and Meldal 10 in 2002. The CuAAC reaction
65

is an ideal candidate for surface coupling because it is selective, quantitative, and
proceeds rapidly in aqueous and mixed aqueous organic solutions under ambient
laboratory conditions. 11 The sole product of the CuAAC reaction is a stable 1,4-
disubstituted 1,2,3-triazole.
Before using the CuAAC reaction for surface coupling, terminal azide or
alkyne functional groups must be introduced onto the surface. We deliberately chose
to introduce azide functional groups onto the surface because azide groups can be
probed relatively easily using Fourier transform infrared spectroscopy (FTIR)
spectroscopy 12 as well as x-ray photoelectron spectroscopy (XPS). 13 The versatility of
the CuAAC reaction on azide-terminated surfaces has been demonstrated through the
attachment of a variety of alkyne-terminated species. 14,15,16,17,18,19,20,21,22,23,24,25 Azide-
terminated siloxane monolayers have often been formed by the solution deposition of
bromide-terminated monolayers followed by SN2 substitution of the bromide groups
with azide groups. 26 This substitution requires long reaction times (~48 h) to approach
completion and sometimes leads to mixtures of bromide and azide functional groups
on the surface. 27,28
To avoid this problem, we have synthesized and purified 11-
azidoundecyltrimethoxysilane as a preformed azide-terminated adsorbate for vapor
deposition onto silicon oxide surfaces. The primary advantage of vapor deposition
over solution deposition is to eliminate the formation of polymeric siloxanes in the
phase contacting the surface. The reduced contamination of surfaces with particles of
these siloxane polymers has been demonstrated using vapor deposition compared to
solution deposition. 29,30,31 We recently reported a method for deposition of dense,
66

methyl-terminated siloxane monolayers onto oxide surfaces from
alkyltrimethoxysilane and water vapors and showed that incomplete monolayers were
deposited without both sufficient silane and sufficient water present. 32 That method is
used to deposit azide-terminated monolayers in this study. Though the vapor
deposition of siloxane monolayers presenting other reactive, terminal functional
groups such as bromide 16 and alkyne 17 groups has been reported, the vapor deposition
of azide-terminated siloxane monolayers has not been reported to our knowledge.
Dilution of the azide groups as one component of a mixed monolayer has
several advantages. Among these is the ability to sufficiently dilute the azide groups to
overcome potential steric limitations in the coupling of species to all azide groups on
the surface. 33 Control over the dilution of the azide groups also allows one to tune the
spacing of and limit the interactions between the species once coupled to the surface.
Mixed siloxane monolayers are usually deposited by coadsorption from organic
solutions containing more than one silane reactant. 34,35,27 The coadsorption of two
silanes from the vapor phase is studied in this paper. The surface compositions of
mixed monolayers are generally different than the initial adsorbate mixtures from
which they were deposited. 36,37,28,38 Therefore, the ability to probe the surface
compositions of mixed monolayers is important, and the azide group provides such a
spectroscopic handle. 37,39
In this paper, we show that denser azide-terminated monolayers are vapor
deposited with a deliberate water source, MgSO4·7H2O, present than without it. Mixed
monolayers are deposited from the vapors of 11-azidoundecyltrimethoxysilane and
alkyltrimethoxysilanes with the water source present. We observe enrichment of the
67

mixed monolayers with the higher vapor pressure silane reactant. It is inferred that the
composition of the mixed monolayers is determined by the composition of the silane
vapor in contact with the oxide surface rather than the remote, liquid silane source.
The quantitative CuAAC reaction of the azide groups in a mixed monolayer and 4-
ethynyl-α,α,α-trifluorotoluene is demonstrated; whereas, the quantitative CuAAC
reaction of a dense monolayer presenting only azide groups is presumably prevented
by steric hindrance.
azide
species of interest
alkyne
Cu(I) catalyst
Cu-catalyzed azidealkyne
cycloaddition
Oxide Surface Oxide Surface
Figure 1. Modular strategy for the covalent modification of oxide surfaces using
mixed, azide-terminated siloxane monolayers followed by the Cu-catalyzed azide-
alkyne cycloaddition (CuAAC) reaction with terminal alkynes to attach species of
interest (red circles).
68

Experimental
Trimethoxysilane Reactants
Decyltrimethoxysilane and hexyltrimethoxysilane were purchased from Alfa-
Aesar and used as received.
11-azidoundecyltrimethoxysilane was prepared from 11-
bromoundecyltrimethoxysilane (Gelest) via displacement of the bromides with sodium
azide following published procedures. 40,41 1.00 g (15 mmol) of sodium azide (Sigma-
Aldrich) was added to a solution containing 2.00 g (5.6 mmol) of 11-
bromoundecyltrimethoxysilane (Gelest) in 30 mL of dry dimethylformamide (Acros).
The solution was allowed to stir for 24 h at room temperature under a nitrogen
atmosphere and then filtered. The filtrate was extracted 3 times with dry pentane. The
pentane was then removed by rotary evaporation. The crude product was purified by
two successive vacuum distillations. CAUTION: Because azide groups can
decompose violently to release nitrogen gas, we limited the mass of the azidosilane to
no more than 2 g and the temperature of the oil bath heat source to no more than
150°C behind a blast shield. In addition, only about 90% of the azidosilane in the pot
was distilled to avoid distilling to dryness. See Figure S1 in the Supporting
Information for a nuclear magnetic resonance (NMR) spectrum.
Tetradecyltri(deuteromethoxy)silane was prepared as previously reported. 32
Surface Cleaning
Single or double-side polished Si(100) wafers, either newly received or after
prior experiments, were cleaned in an oxygen plasma cleaner (Harrick) for 10 min at
medium power with a dioxygen flow rate of 50 std. mL/min at a pressure of 250
69

mTorr. This cleaning method resulted in hydrophilic surfaces with water contact
angles approaching 0°. 42 Surfaces were exposed to atmospheric air for up to 15 min
after cleaning while determining effective refractive indices of the substrates by
ellipsometry.
Monolayer Vapor Deposition
Siloxane monolayers were vapor-deposited onto silicon oxide surfaces in o-
ring sealed, glass vacuum desiccators fitted with Teflon stopcocks (Jencons part
number 250-048) with internal volumes of about 600 mL. These chambers were oven
dried for a minimum of 4 h at 140°C in air and cooled on the bench top for no more
than 10 min in air at room temperature before use. Neat silane (100 µL), either pure
azidosilane or a silane mixture, was pipetted onto and absorbed into 42.5 mm diameter
Whatman filter paper dried in the same way in the bottom of the desiccator. 0.5 g of
MgSO4·7H2O (Fisher biochemical grade) were placed in a foil boat in the bottom of
the desiccator to serve as a water source for the hydrolysis reaction. The plasma-
cleaned surfaces were placed on a metal rack in the chamber above the silane liquid
and hydrated salt. The desiccator was then evacuated through a rubber hose to a glass
vacuum line with a liquid nitrogen-trapped mechanical pump for approximately 60
seconds. The final pressure at the trap was 1 Torr. The Teflon valve on the desiccator
was closed, and the chamber was placed in a 110°C preheated oven (Forma Scientific)
for various periods of time from 1 to 24 h. After deposition, the valve was opened to
ambient air to return the chamber to atmospheric pressure and to remove the samples.
The following mixtures were used for the mixed monolayer depositions: 0.250 g
azidosilane and 0.620 g decyltrimethoxysilane (χ N3 ,liquid = 0.25), 0.500 g azidosilane
70

and 0.413 g decyltrimethoxysilane (χ N3 ,liquid = 0.50), 0.750 g azidosilane and 0.207 g
decyltrimethoxysilane (χ N3 ,liquid = 0.75), 0.750 g azidosilane and 0.162 g
hexyltrimethoxysilane (χ N3 ,liquid = 0.75), 0.750 g azidosilane and 0.258 g
tetradecyltri(deuteromethoxy)silane (χ N3 ,liquid = 0.75).
Surface Characterization
FTIR spectra were obtained with a Bruker Vertex 70 spectrometer using a KBr
beam splitter and a deuterated triglycine sulfate (DTGS) detector. Spectra were
collected in transmission mode through the silicon with the samples at the silicon
Brewster‟s angle (74°) using p-polarized light from a wire-grid polarizer (Specac
KRS-5). For each spectrum, 1024 scans were collected at 4 cm -1 resolution.
Background spectra were collected from freshly oxygen plasma-cleaned, double side
polished Si(100) crystals in the same orientation.
Ellipsometry measurements were performed using a Gaertner L116
ellipsometer at 70° angle of incidence. Effective real and imaginary refractive indices
of the Si(100) substrates were determined immediately after plasma cleaning but
before siloxane monolayer vapor deposition. Measurements after vapor deposition,
rinsing with toluene and isopropyl alcohol, and drying in a stream of nitrogen were
used to determine monolayer thicknesses. A film refractive index of 1.46 was used for
all thickness calculations. At least three measurements were performed on each
sample to obtain average substrate refractive indices or sample thicknesses.
Advancing sessile drop water contact angles were measured with a Ramé-Hart
model 100 goniometer using water from a four-bowl Millipore purification system.
71

Water droplets were dispensed from a syringe with a flat-tipped needle. Fresh water
was obtained from the Millipore system for each measurement session. At least two
measurements were made on each sample on both sides of the droplets and averaged.
X-ray photoelectron spectroscopy (XPS) was performed using a Physical
Electronics Inc. 5000 Versa Probe spectrometer with Al Kα radiation (1486.6 eV).
High-resolution spectra were the result of the co-addition of 10 scans collected at a
step size of 0.1 eV, an analyzer pass energy of 117 eV, and a takeoff angle of 45°.
Cu-Catalyzed Azide-Alkyne Cycloaddition
CuAAC reactions were performed by exposing azide-terminated monolayers to
3:2 dimethyl sulfoxide (DMSO):H2O solutions containing 1 mM 4-ethynyl-α,α,α-
trifluorotoluene, 500 µM CuSO4·5H2O, 500 µM of 1,1',1''-Tris(1H-1,2,3-triazol-4-yl-
1-acetic acid ethyl ester) trimethylamine (TTMA; CAS#736958-00-2; see Supporting
Information for structure), and 5 mM sodium ascorbate for various periods of time.
Next, the surfaces were rinsed with chloroform, toluene, isopropyl alcohol, water, and
isopropyl alcohol again. The surfaces were then sonicated for 1 minute in chloroform
and rinsed with isopropyl alcohol and dried in a stream of nitrogen. TTMA was
synthesized following a published procedure. 43
Results
Azide Functional Group as a Spectroscopic Handle
The ability to monitor the azide groups on surfaces provides a way to follow
the progress of the CuAAC reaction. Figure 2 shows a p-polarized Brewster‟s angle
transmission FTIR spectrum of a monolayer vapor deposited from the azidosilane for
12 h with 0.5 g MgSO4·7H2O present. The peak at 2100 cm -1 is assigned to the
72

asymmetric stretching mode of the azide group, 44 which can be used to monitor
relative azide surface compositions. In addition to the azide absorbance, two other
peaks are present at 2855 cm -1 and 2925 cm -1 . These peaks correspond to the
symmetric and asymmetric methylene stretching modes of the alkyl chains in the
monolayer. 45
Figure 2. Azide asymmetric and carbon-hydrogen stretching regions of the p-
polarized Brewster‟s angle transmission FTIR spectrum of a χ N3 ,liquid = 1.0 monolayer
on silicon oxide after a 12 h vapor deposition from 100 µL of 11-
azidoundecyltrimethoxysilane with 0.5 g MgSO4·7H2O present.
XPS can also be used to monitor the presence of azide groups on the surface.
In the case of azide groups, the XPS spectrum yields distinct bonding information in
the N 1s region. As shown in Figure 3, an asymmetric doublet is present in the high-
resolution N 1s XPS spectrum of an azide-terminated monolayer that was vapor
deposited onto silicon oxide. The higher binding energy XPS peak is assigned to the
73

electron-deficient, central nitrogen atom in the azide group, while the lower binding
energy peak is assigned to the two outer nitrogen atoms in the azide group as shown
by the peak fitting. 46 Figure 3 also shows the Si 2p photoelectron region. The lower
binding energy peak is assigned to the bulk silicon atoms in the substrate, and the
higher binding energy peak is assigned to the silicon atoms bonded to electron-
withdrawing oxygen atoms. The ratios of the integrated areas of the N 1s and Si 2p
peaks are used to probe the relative azide surface compositions after vapor depositions
and after CuAAC reactions.
Figure 3. High-resolution Si 2p (95 - 110 eV) and N 1s (395 - 410 eV) XPS spectra
for a χ N3 ,liquid = 1.0 monolayer on silicon oxide after 12 h of vapor deposition from 100
µL of 11-azidoundecyltrimethoxysilane with 0.5 g MgSO4·7H2O present. The
asymmetric doublet characteristic of the azide group in the high-resolution N 1s
spectrum is fitted by three Gaussian peaks, one for each nitrogen atom in the azide
group.
74

Time Dependence of Monolayer Formation
Figure 4a shows the time dependence of ellipsometric thicknesses measured on
monolayers deposited at 110°C from the vapor of 100 µL of 11-
azidoundecyltrimethoxysilane without and with 0.5 g MgSO4·7H2O present in the
deposition chamber. Larger thicknesses are observed for the monolayers deposited
with MgSO4·7H2O. Limiting ellipsometric thicknesses are observed after 12 h in both
cases. The limiting thickness without 0.5 g MgSO4·7H2O is 12.1 Å; whereas, it is 14.4
Å with 0.5 g MgSO4·7H2O.
Figure 4b is a plot of the integrated azide absorbance (Figure 2) versus
deposition time. The azide integrated absorbance increases with increasing deposition
time until reaching a limiting value after about 12 h of deposition. The azide
absorbance is higher for depositions performed in the presence of MgSO4·7H2O than
for depositions without MgSO4·7H2O.
Figure 4c is a plot of the ratio of the areas of the N 1s and Si 2p XPS peaks
(Figure 3) as a function of time. The N 1s:Si 2p ratios increase with time and are
higher at all time points for depositions performed with 0.5 g MgSO4·7H2O than for
those without. Limiting values are reached after about 12 h of deposition.
The time dependence of the water contact angle is plotted in Figure 4d for
depositions with and without 0.5 g MgSO4·7H2O. The water contact angles increase
with increasing deposition time before reaching the same limiting value after 4 h. No
difference in the limiting values of the water contact angles is observed between
depositions performed with or without MgSO4·7H2O. This is in contrast to our work
75

with methyl-terminated monolayers where larger water contact angles were measured
with 0.5 g MgSO4·7H2O than without it. 32
76

Ellipsometric Thickness / Å
Integrated Azide Absorb. / cm -1
XPS N 1s:Si 2p
Ellipsometric Thickness / Å
Water Contact Angle / °
0.04
0.03
0.02
0.01
0
0.45
0.30
0.15
16
0
16
12
8
4
0
12
60
80 (d)
8
40
4 20
0
0
(a)
(b)
(c)
without MgSO 4 ·7H 2 O
with 0.5 g MgSO 4 ·7H 2 O
with 0.5 g MgSO 4 ·7H 2 O
without MgSO 4 ·7H 2 O
0 0 55 10 15 20 20 25 25
77
Time / h
Figure 4. Time dependence of vapor deposition of χ N3 ,liquid = 1.0 monolayers onto
silicon oxide surfaces from 100 µL of 11-azidoundecyltrimethoxysilane with and

without 0.5 g MgSO4·7H2O present. The time dependence was monitored using (a)
ellipsometry, (b) FTIR spectroscopy, (c) XPS, and (d) water contact angle goniometry.
CuAAC Reactions on Dense, Azide-Terminated Monolayers
Once conditions for vapor depositing dense, azide-terminated monolayers were
established, the reactivity of the azide groups with terminal alkynes using the CuAAC
reaction was assessed using FTIR spectroscopy. Figure 5a shows FTIR spectra of a
pure azidosilane monolayer before and after a 1 h exposure to a solution containing 4-
ethynyl-α,α,α-trifluorotoluene and the Cu(I) catalyst for the CuAAC reaction. After 1
h of reaction, the intensity of the azide stretch is reduced but is still observable. In
addition, a new peak at 1328 cm -1 is present in the spectrum, which is assigned to the
asymmetric C-F stretching mode. 37,47,48 The inset in Figure 5a shows the time
dependence of the azide integrated absorbance during the CuAAC reaction with 4-
ethynyl-α,α,α-trifluorotoluene. The azide absorbance decreases with increasing time
until reaching a minimum limiting value after 1 h of reaction. Increasing the reaction
time to 6 h did not result in any further decrease of the azide absorbance.
78

Figure 5. Carbon-fluorine and azide asymmetric stretching regions of the p-polarized
Brewster‟s angle transmission FTIR spectrum of (a) χ N3 ,liquid = 1.0 and (b) χ N3 ,liquid =
0.75 monolayers on silicon oxide before and after 1 h of a CuAAC reaction with 4-
ethynyl-α,α,α-trifluorotoluene. The monolayers were vapor-deposited for 12 h from
100 µL of liquid silane with 0.5g MgSO4·7H2O present. The inset in (a) shows the
time dependence of the integrated azide FTIR absorbance during CuAAC reactions
between 4-ethynyl-α,α,α-trifluorotoluene and χ N3 ,liquid = 1.0 monolayers on silicon
oxide.
79

Mixed Monolayers
Mixed monolayers were vapor deposited from azidosilane and
alkyltrimethoxysilane liquid sources of known composition and 0.5 g MgSO4·7H2O.
Figure 6a shows the integrated azide absorbance versus the number fraction of the
azidosilane (χ N3 ,liquid) in the liquid silane source. The azide absorbance monotonically
increases with increasing χ N3 ,liquid. The straight, dashed line labeled with a „1‟
interpolates between the integrated absorbance for χ N3 ,liquid = 0, where no azide
absorbance is observed, and χ N3 ,liquid = 1, where maximum azide absorbance is
observed. With decyltrimethoxysilane as the diluent (circles), the integrated azide
absorbances are all less than the straight, dashed line.
The surface compositions of the mixed monolayers are also assessed using N
1s:Si 2p XPS ratios (Figure 6b). Increasing χ N3 ,liquid results in an increase in the N
1s:Si 2p ratio until reaching the maximum at χ N3 ,liquid = 1. The N 1s:Si 2p ratios for all
monolayers deposited from mixtures of the azidosilane and decyltrimethoxysilane
(circles) are less than the straight, dashed line that interpolates between the two
limiting values. Figure 6c shows the dependence of the water contact angles on
χ N3 ,liquid for monolayers deposited from mixtures of the azidosilane and
decyltrimethoxysilane. The contact angles range from 80° for χ N3 ,liquid = 1 to 109° for
χ N3 ,liquid = 0.
The effect of varying the alkyl chain length of the alkyltrimethoxysilane
diluent was explored for χ N3 ,liquid = 0.75 using FTIR spectroscopy and XPS.
80

Monolayers vapor deposited from mixtures of the azidosilane and
hexyltrimethoxysilane exhibit lower azide integrated absorbances and lower N 1s:Si
2p XPS ratios (Figures 6a and b, diamonds) than those deposited from mixtures of the
azidosilane and decyltrimethoxysilane. Larger azide absorbances and N 1s:Si 2p ratios
(Figures 6a and b, squares) are measured with tetradecyltrimethoxysilane as the
diluent compared to decyltrimethoxysilane.
81

Integrated azide absorbance / cm -1
water contact angle / °
XPS N 1s: Si 2p
0.04
0.02
0
0.4
0.2
0
110
100
90
80
(a)
(b)
(c)
C14 diluent
C10 diluent
C6 diluent
0 0.25 0.50
N3 ,liquid
0.75 1.00
Figure 6. Relative surface compositions of mixed monolayers vapor deposited from
the azidosilane and alkyltrimethoxysilane diluents as inferred from: (a) integrated
azide FTIR absorbances, (b) N 1s:Si 2p XPS ratios, and (c) water contact angles. The
82
1
2
5
10
20
50
1
2
5
10
20
50

C6, C10, and C14 alkyltrimethoxysilane diluents are hexyltrimethoxysilane
decyltrimethoxysilane, and tetradecyltri(deuteromethoxy)silane, respectively. χ N3 ,liquid
is the mole fraction of 11-azidoundecyltrimethoxysilane in the liquid silane source
used during the vapor depositions. The monolayers were vapor-deposited for 12 h
from 100 µL of the silane mixtures with 0.5 g MgSO4·7H2O present. The dashed and
dotted lines in (a) and (b) represent Raoult‟s law predictions for various vapor pressure
ratios of the diluent silane to the azidosilane. 49 The numbers 1, 2, 5, 10, 20 and 50
represent the diluent silane to azidosilane vapor pressure ratios for each of the Raoult‟s
law predictions. The solid line in (c) is a guide to the eye.
CuAAC Reactions on Mixed Monolayers
Figure 5b shows the azide asymmetric stretching region of FTIR spectra
measured on a χ N3 ,liquid = 0.75 monolayer with a decyltrimethoxysilane diluent before
and after a 1 h CuAAC reaction with 4-ethynyl-α,α,α-trifluorotoluene. The azide
absorbance at 2100 cm -1 is reduced to the level of noise after 1 h of reaction,
confirming quantitative conversion of the azide groups to triazoles.
XPS is also used to monitor changes on the surface after the CuAAC reaction.
Figure 7c is a high-resolution XPS spectrum of the N 1s region measured on a χ N3 ,liquid
= 0.75 monolayer with a decyltrimethoxysilane diluent, and the asymmetric doublet
characteristic of the azide group is observed. After 1 h of a CuAAC reaction with 4-
ethynyl-α,α,α-trifluorotoluene, the higher binding energy azide peak is no longer
83

present (Figure 7d). Instead, one broad peak at 401 eV is observed and is assigned to
the three nitrogen atoms in the 1,2,3-triazole product of the CuAAC reaction.
In addition to the disappearance of the higher binding energy N 1s peak after
the CuAAC reaction, changes are observed in other regions of the XPS spectrum and
provide further evidence for the covalent attachment of 4-ethynyl-α,α,α-
trifluorotoluene. After the CuAAC reaction, a peak is observed in the F 1s region at
689 eV, assignable to the fluorine atoms in the trifluoromethyl group as shown in
Figure 7f, while no peak is observed in the spectrum of the mixed monolayer before
reaction (Figure 7e). 37 A small peak at 293 eV, assignable to the electron-deficient
carbon atom in the trifluoromethyl group, is present in the C 1s spectrum after reaction.
The lower binding energy C 1s peak at 285 eV that is present before and after reaction
is assigned to the other carbon atoms in the monolayer.
84

Figure 7. High-resolution XPS spectra of the C 1s [(a) and (b)], N 1s [(c) and (d)], and
F 1s [(e) and (f)] photoelectron regions for a χ N3 ,liquid = 0.75 monolayer on silicon
oxide before and after 1 h of a CuAAC reaction with 4-ethynyl-α,α,α-trifluorotoluene.
The monolayer was vapor-deposited for 12 h from 100 µL of a χ N3 ,liquid = 0.75 mixture
of 11-azidoundecyltrimethoxysilane and decyltrimethoxysilane with 0.5 g
MgSO4·7H2O present. The N 1s spectra are fitted by three Gaussian peaks, one for
each nitrogen atom in the azide group, as shown in panels (c) and (d).
Discussion
Mixed, azide-terminated siloxane monolayers provide an ideal platform for the
covalent modification of oxide surfaces with a variety of substituents because the
azide composition is reproducible and can be conveniently probed using FTIR
spectroscopy and XPS.
85

Effect of Water Addition
With MgSO4·7H2O present as a water source during vapor deposition, a
higher coverage of the monolayers is obtained as inferred from the larger limiting
values of the integrated azide FTIR absorbances and N 1s: Si 2p XPS ratios compared
to monolayers deposited without MgSO4·7H2O (Figure 4). The methylene stretching
modes in the 2800-3000 cm -1 range provide further evidence that complete
monolayers are deposited with MgSO4·7H2O present. The positions of the symmetric
and asymmetric methylene stretches shift to lower frequency as the packing of alkyl
chains becomes denser. 50,51 The observation of the symmetric and asymmetric
methylene FTIR stretching modes at 2855 and 2925 cm -1 for monolayers vapor-
deposited for 12 h from the azidosilane with MgSO4·7H2O present is consistent with
previous reports for the solution deposition of dense azide 52 and methyl-terminated 53
siloxane monolayers with 11-carbon alkyl chains from trichlorosilanes.
In addition, larger ellipsometric thicknesses are observed on monolayers vapor
deposited with MgSO4·7H2O present than without it. The thicknesses we observe with
MgSO4·7H2O present, 14.4 Å, are comparable to literature values 52,23 that range from
13.5 Å to 14.8 Å for monolayers solution-deposited from bromoundecyltrichlorosilane
followed by SN2 displacement of the bromide groups on the surface with azide groups.
We infer that complete monolayers are vapor deposited with MgSO4·7H2O present
and that incomplete monolayers are deposited without MgSO4·7H2O. We have
previously observed a similar difference in ellipsometric thicknesses for monolayers
vapor-deposited from tetradecyltri(deuteromethoxy)silane with and without 0.5 g
MgSO4·7H2O present and found that unhydrolyzed methoxy groups remained on
86

adsorbed silanes without sufficient MgSO4·7H2O. 32 Although the larger azide
absorbances, N 1s:Si 2p ratios, and ellipsometric thicknesses observed with
MgSO4·7H2O present are qualitatively consistent, the increase in the azide absorbance
and N 1s:Si 2p ratio with MgSO4·7H2O present is larger than the increase observed in
the ellipsometric thickness. We do not have an explanation for this discrepancy.
CuAAC Reactions on Pure and Mixed Azide-Terminated Monolayers
The residual azide absorbance observed for χ N3 ,liquid = 1 monolayers after long
exposure to CuAAC reaction conditions (Figure 5a) with 4-ethynyl-α,α,α-
trifluorotoluene can be attributed to steric crowding on the surface. Devaraj et al.
found that steric crowding prevents the quantitative reaction of the azide groups in
dense monolayers deposited from azide-terminated thiols. 39 To overcome steric
crowding on the surface, mixed monolayers were vapor deposited to dilute the azide
functionality with unreactive methyl groups. The quantitative reaction of the azide
groups was obtained with azide surface coverages up to approximately 40 percent as
inferred from the disappearance of the azide absorbance in the FTIR spectrum (Figure
5b) and from the disappearance of the higher binding energy azide peak in the high-
resolution N 1s XPS spectrum after 1 h of reaction. This quantitative reaction of the
azide groups at relatively high azide surface coverage suggests that uniform mixing of
the azide and methyl-terminated silanes, as opposed to segregation, is occurring in the
mixed monolayers.
Tuning the Azide Surface Composition with Mixed Monolayers
When the azide groups are sufficiently isolated on the surface for quantitative
CuAAC reactions, knowledge of the relative azide surface composition permits the
87

predictable attachment of an alkyne-terminated species at a desired relative azide
surface composition. FTIR spectroscopy, XPS, and contact angle goniometry tell us
that the vapor-deposited mixed monolayers are enriched with methyl-terminated alkyl
chains from the decyltrimethoxysilane diluent because the azide integrated
absorbances and N 1s: Si 2p XPS ratios for the mixtures are less than the dashed lines
(Figures 6a and b, circles), which represent surface compositions equal to χ N3 ,liquid. The
water contact angles also suggest enrichment of the unsubstituted
decyltrimethoxysilane on the surface. Why are the surface compositions different than
χ N3 ,liquid?
We hypothesize that the relative azide surface composition is the same as the
composition of the vapor that contacts the surface. We further hypothesize that the
vapor is in equilibrium with the liquid silane source and that Raoult‟s law predicts the
composition of the vapor in equilibrium with the liquid mixture. Predictions for
integrated azide absorbances and N 1s:Si 2p XPS ratios from vapor compositions
based upon Raoult‟s law are represented in Figures 6a and b by the dotted and dashed
lines. 49 Predictions are plotted for vapor pressure ratios of the n-alkyltrimethoxysilane
diluent to the azidosilane from 1 to 50. With decyltrimethoxysilane as the diluent
(circles), both the FTIR data and the XPS data approximately resemble the Raoult‟s
law prediction for a 5:1 vapor pressure ratio of the diluent to the azidosilane. A larger
vapor pressure ratio of approximately 10 is expected by comparing the ratios of the
vapor pressures of n-alkanes with comparable molecular weights to the silanes, 54,55,56
suggesting that more enrichment of the monolayer with the decyltrimethoxysilane
diluent is expected than obtained from experiment. Perhaps the adsorption of the
88

longer chain azidosilane is slightly preferred over the shorter decyltrimethoxysilane
diluent. 36 Experiments in which the alkyl chain length, and therefore vapor pressure,
of the alkyltrimethoxysilane diluent was varied (Figures 6a and b) result in the
expected trend. When the higher vapor pressure hexyltrimethoxysilane is used as the
diluent, a greater enrichment of methyl functionality on the surface is observed
compared to decyltrimethoxysilane; whereas, a lesser enrichment of methyl
functionality is obtained when the lower vapor pressure tetradecyltrimethoxysilane
diluent is used as inferred from integrated azide absorbances and N 1s:Si 2p ratios.
Conclusions
Pure and mixed azide-terminated siloxane monolayers were vapor deposited
onto silicon oxide surfaces from 11-azidoundecyltrimethoxysilane and n-
alkyltrimethoxysilane diluents. The Cu-catalyzed azide-alkyne cycloaddition
(CuAAC) reaction was used to covalently attach an alkyne-terminated species, 4-
ethynyl-α,α,α-trifluorotoluene, to the azide-terminated surfaces. The azide group is a
sensitive spectroscopic probe for surface analysis using FTIR spectroscopy and XPS
to monitor relative azide surface compositions. Denser azide-terminated monolayers
were achieved from depositions with 0.5 g MgSO4·7H2O present as a water source as
inferred from the larger ellipsometric thicknesses, integrated azide FTIR absorbances,
and N 1s:Si 2p XPS ratios that were measured with MgSO4·7H2O than without it.
The relative azide surface compositions in mixed monolayers were not equal to
the initial, liquid silane compositions. Enrichment of the higher vapor pressure silane
reactant was observed in the mixed monolayers and was attributed to the difference
between the silane vapor composition in contact with the surface and the remote liquid
89

source composition. Quantitative reaction of the azide groups on the surface was
achieved on mixed monolayers with the azide functionality diluted to a surface
composition of approximately 40% with unreactive methyl groups, which suggests
uniform mixing of the two components in the monolayers. Steric constraints
presumably prevented the quantitative reaction of the azide groups in the pure azide-
terminated monolayers.
Acknowledgments
This work was supported by funding from Helicos BioSciences Corporation. The
authors acknowledge NSF grant CHE-0639053 for support of FTIR equipment. The
authors also acknowledge NSF grant DMR-0213618 to the Center on Polymer
Interfaces and Macromolecular Assemblies for use of the ellipsometer. Useful
discussions with Dr. Erin Artin, Dr. Tim Harris, and Dr. Neal Devaraj are
acknowledged.
90

Supporting Information
Figure S1. NMR spectrum of the synthesized 11-azidoundecyltrimethoxysilane used
for siloxane monolayer vapor depositions. The spectrum was obtained on a 300 MHz
Varian Inova spectrometer in CDCl3 solvent.
91

Figure S2. Chemical structure drawing of TTMA, the copper ligand used for CuAAC
reactions.
92

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33. Guo, F.; Jankova, K.; Schulte, L.; Vigild, M. E.; Ndoni, S. Langmuir 2009, 26,
2008-2013.
34. Fadeev, A. Y.; McCarthy, T. J. Langmuir 1999, 15, 7238-7243.
35. Offord, D. A.; Griffin, J. H. Langmuir 2002, 9, 3015-3025.
36. Bain, C. D.; Whitesides, G. M. Journal of the American Chemical Society
1989, 111, 7164-7175.
37. Azam, M. S.; Fenwick, S. L.; Gibbs-Davis, J. M. Langmuir 2011, 27, 741-750.
95

38. Tong, Y.; Tyrode, E.; Osawa, M.; Yoshida, N.; Watanabe, T.; Nakajima, A.;
Ye, S. Langmuir 2011, 27, 5420-5426.
39. Collman, J. P.; Devaraj, N. K.; Eberspacher, T. P. A.; Chidsey, C. E. D.
Langmuir 2006, 22, 2457-2464.
40. Fu, Y.-S.; Yu, S. J. Angewandte Chemie (International ed.) 2001, 40, 437-440.
41. Alauzun, J.; Besson, E.; Mehdi, A.; Rey; xe; Catherine; Corriu, R. J. P. Chem.
Mater. 2008, 20, 503-513.
42. Some of the surfaces were subsequently cleaned in a solution consisting of 10
mL of 30% hydrogen peroxide (Kanto) and 10 mL of 29% ammonium hydroxide
(Kanto) in 60 mL Millipore water 70°C. Surfaces were rinsed copiously with
deionized water and then dried by rinsing with isopropanol and blow drying in a
stream of nitrogen. The surfaces were then subjected to another oxygen plasma clean
under the same conditions before using them. No difference in the resulting
monolayers was observed between monolayers that were deposited onto surfaces that
were only plasma cleaned and monolayers deposited onto surfaces that were subjected
to the additional basic solution and plasma cleaning steps. .
43. Zhou, Z.; Fahrni, C. J. JACS 2004, 126, 8862-8863.
44. Lieber, E.; Rao, C. N. R.; Chao, T. S.; Hoffman, C. W. W. Analytical
Chemistry 1957, 29, 916-918.
45. Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. Journal of the
American Chemical Society 1987, 109, 3559-3568.
46. Wollman, E. W.; Kang, D.; Frisbie, C. D.; Lorkovic, I. M.; Wrighton, M. S.
Journal of the American Chemical Society 1994, 116, 4395-4404.
96

47. Nomaru, K.; Gorelik, S. R.; Kuroda, H.; Nakai, K. Nuclear Instruments and
Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and
Associated Equipment 2004, 528, 619-622.
48. Qu, L.; Xin, Z. Langmuir 2011, 27, 8365-8370.
49. The dotted and dashed lines in Figure 6 are predictions of the silane vapor
mixture composition based upon Raoult‟s law for various ratios of the vapor pressure
of the n-alkyltrimethoxysilane diluent (Pvapor,diluent) to the vapor pressure of the
azidosilane (Pvapor,N 3 ). The predicted mole fraction of the azidosilane in the vapor
phase ( χ N 3 ,vapor) was then multiplied by the integrated azide absorbance and N 1s:Si 2p
XPS ratio measured for monolayers vapor deposited from the pure azidosilane
( χ N 3 ,liquid = 1) to convert the predicted vapor composition to an equivalent predicted
surface composition. The Raoult‟s law equation used to make the predictions is:
P
N , liquid
vapor,
N
3
3
N , vapor
3
P 1
P
N , liquid vapor,
N N , liquid
vapor,
diluent
3
3 3
50. Snyder, R. G.; Strauss, H. L.; Elllger, C. A. J. Phys. Chem. 1982, 86, 5145-
5150.
51. Snyder, R. G.; Maroncelli, M.; Strauss, H. L.; Hallmark, V. M. The Journal of
Physical Chemistry 1986, 90, 5623-5630.
52. Lummerstorfer, T.; Hoffmann, H. J. Phys. Chem. B 2004, 108, 3963-3966.
53. Hoffmann, H.; Mayer, U.; Krischanitz, A. Langmuir 1995, 11, 1304-1312.
97

54. The expected ratio of 10:1 for the vapor pressures of decyltrimethoxysilane
(MW = 262.5) to azidoundecyltrimethoxysilane (MW = 317.5) results from a
comparison between nonadecane (MW = 268.5) with a vapor pressure of 0.37 Torr
and tricosane (MW = 324.6) with a vapor pressure of 0.033 Torr at the same
temperature used for the vapor depositions, 110°C. These vapor pressures were
calculated from Antoine equation parameters available on the online NIST
WebBook. 55 Literature reports for the vacuum distillation of decyltrimethoxysilane 56
and azidoundecyltrimethoxysilane 31 near 110°C suggest a larger vapor pressure ratio
of approximately 70. Vacuum distillations are often performed under dynamic vacuum
and the specific details are often omitted in synthetic preparations, which may
invalidate the assumption that a reported distillation pressure is an accurate
measurement of vapor pressure.
55. P.J. Linstrom and W.G. Mallard, Eds., NIST Chemistry WebBook, NIST
Standard Reference Database Number 69, National Institute of Standards and
Technology, Gaithersburg MD, 20899, http://webbook.nist.gov, (retrieved May 2011).
56. Ameduri, B.; Boutevin, B.; Moreau, J. J. E.; Moutaabbid, H.; Chi Man, M. W.
Journal of Fluorine Chemistry 2000, 104, 185-194.
98

Chapter 4: Attachment and Hybridization of Oligonucleotides on Azide-Terminated
Siloxane Monolayers with Resistance to Nonspecific Adsorption
Preface
This chapter of my dissertation is derived from a manuscript that may be
submitted to a scientific journal for publication. I am the first author of this manuscript
and am responsible for most of the writing and preparation. The other authors are Dr.
Erin Artin, David G. Lapham, Dr. Timothy D. Harris, and Prof. Christopher E. D.
Chidsey. My contributions to this work include: vapor deposition of azide-terminated
monolayers onto glass and silicon oxide, synthesis of 11-
azidoundecyltrimethoxysilane and ethynyl phosphonic acid, bulk fluorescence
measurements on glass surfaces, optimization of copper-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction conditions, and acquisition of X-ray photoelectron
spectra.
Dr. Artin synthesized the alkyne-terminated oligonucleotide and Cy3-alkyne
dye and contributed to the optimization of CuAAC conditions. She also performed the
single molecule fluorescence measurements. David Lapham performed the
hybridization experiments shown in Figure 4. Dr. Harris and Prof. Chidsey originally
proposed the idea to use the CuAAC reaction to attach oligonucleotides to azide-
terminated monolayers on glass surfaces for single molecule sequencing by synthesis
applications and contributed intellectually by providing guidance throughout the
progression of this work. Prof. Chidsey, Dr. Harris, and Dr. Artin also assisted in the
preparation and the writing of the manuscript.
99

Abstract
A method is presented to covalently attach dye-labeled oligonucleotides to
glass surfaces followed by hybridization with complementary oligonucleotides with
minimal nonspecific adsorption. Azide-terminated siloxane monolayers are first vapor
deposited onto glass surfaces and are found to be susceptible to the nonspecific
adsorption of oligonucleotides. The nonspecific adsorption is reduced after the
attachment of ethynyl phosphonic acid to the surface using the copper-catalyzed azide-
alkyne cycloaddition (CuAAC) reaction. A two-step CuAAC reaction sequence is
used to first attach Cy3-labeled, alkyne-terminated oligonucleotides followed by a
second CuAAC reaction with ethynyl phosphonic acid as an agent to reduce
nonspecific adsorption. After reaction, complementary oligonucleotides are captured
on the surface by hybridization to the surface-attached oligonucleotides. Bulk and
single-molecule fluorescence measurements are used to characterize the surfaces
before and after exposure to dye-labeled oligonucleotides.
100

Introduction
In an effort to capture oligonucleotide targets by specific hybridization
interactions between complementary nucleotides, a platform was developed to present
covalently attached oligonucleotide probes surrounded by blocking agents to resist
nonspecific adsorption on glass surfaces. The covalent attachment of biochemically
viable oligonucleotides to surfaces is important for many technologies such as next-
generation DNA sequencing and biochemical sensing. 1,2,3 Glass surfaces are attractive
substrates for oligonucleotide immobilization because they are relatively cheap and
compatible with optical measurements such as fluorescence spectroscopy where a low
background fluorescence is desired. 4,5,6
Oligonucleotides can be covalently bound to glass and other oxide surfaces by
either appending an appropriate anchoring group, such as a silane, that will bond with
the surface 7 or by introducing compatible reactive groups to the oligonucleotide and
oxide surface and then allowing them to react. 6 The first method has disadvantages
because tethering an anchoring group to an oligonucleotide may require difficult
syntheses each time one wants to change the oligonucleotide sequence. Furthermore,
the attachment of an anchoring group to a bulky oligonucleotide strand does not
guarantee that the oligonucleotide will reproducibly form a monolayer or be viable for
hybridization with complementary oligonucleotides once adsorbed.
Decoupling the surface attachment of oligonucleotides into an adsorption step
to introduce reactive functionality on the surface followed by a cross-coupling reaction
to covalently attach oligonucleotides allows independent optimization at each step and
is especially advantageous when the cross-coupling chemistry between the surface and
101

oligonucleotides is robust. 8 The copper-catalyzed, azide-alkyne cycloaddition
(CuAAC) reaction has been shown to be a nearly ideal cross-coupling chemistry for
attaching species to surfaces. 9 Because the CuAAC reaction is such an ideal surface
reaction, it has already been used to covalently attach functionalized oligonucleotides
to azide 10,11,12,13
and alkyne-terminated monolayers. 14,15,16 Azide-terminated
monolayers are favored because azide groups can be easily probed using Fourier
transform infrared (FTIR) and x-ray photoelectron spectroscopy (XPS). 17 Azide-
terminated siloxane monolayers can be used to introduce azide functionality onto glass
and other oxide surfaces as previously reported. 18
The use of a dense, hydrophobic monolayer and a selective surface chemistry
such as the CuAAC reaction does not make surfaces immune to a major problem
encountered when attaching biomolecules to them, nonspecific adsorption. Complex
biomolecules such as oligonucleotides often nonspecifically adsorb onto hydrophobic
monolayer surfaces from their bulk solutions upon exposure. 19 This nonspecific
adsorption is undesirable for most applications and should be minimized. Various
methods are used to reduce the nonspecific adsorption of biomolecules such as rinsing
procedures 20 and the introduction of functional groups on the surface such as
polyethylene glycols (PEGs), 21,22,23 acrylamide, 24 polysaccharides, 25,26 or charged
species 27,28,29,30 that can add electrostatic or hydrophilic character to the surface to
resist nonspecific adsorption. A mixed monolayer consisting of oligonucleotides
surrounded by functional groups that can add nonspecific adsorption resistance to the
surface during hybridization may be attainable using two CuAAC reactions as outlined
in Figure 1.
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Fluorescence spectroscopy is a useful technique for monitoring the attachment
of dye-labeled oligonucleotides to surfaces and their subsequent hybridization with
dye-labeled complementary strands. 31,32 Single molecule fluorescence is a particularly
sensitive technique for characterizing surfaces modified with dye-labeled
oligonucleotides, and it has recently been used for sequencing by synthesis
applications. 33,34 One of the problems encountered with performing fluorescence
measurements is producing reactive, optical surfaces with sufficiently low background
fluorescence to easily discern fluorescence attributable to the attachment of a dye-
labeled species such as a biomolecule. 35,36 Aleman et al. recently reported single
molecule fluorescence measurements of Cy3-labeled oligonucleotides that were
covalently attached to solution-deposited, azide-terminated siloxane monolayers on
glass substrates using the CuAAC reaction. 14
In this study, we demonstrate the covalent attachment of alkyne-terminated,
dye-labeled oligonucleotides onto vapor-deposited, azide-terminated siloxane
monolayers using the CuAAC reaction. Bulk and single-molecule fluorescence
spectroscopies are used to characterize the surfaces. The nonspecific adsorption of
dye-labeled oligonucleotides onto the azide-terminated monolayers is reduced by
forming a mixed monolayer of oligonucleotides and ethynyl phosphonic acid groups
using two, sequential CuAAC reactions. This platform is used to capture
complementary oligonucleotides on the surface via specific hybridization to covalently
attached oligonucleotides with minimal nonspecific adsorption.
103

(a) (b)
1. CuAAC
Oxide Surface Oxide Surface
(d) (c)
3. Hybridization
104
2. CuAAC
Oxide Surface Oxide Surface
Figure 1. Schematic of the platform developed for the attaching and hybridizing dye-
labeled oligonucleotides on oxide surfaces. First, an azide-terminated monolayer is

vapor deposited as shown in (a) and then a CuAAC reaction is performed with alkyne-
terminated oligonucleotides which results in covalently attached and nonspecifically
adsorbed oligonucleotides as shown in (b). This is followed by rinsing and a CuAAC
reaction with ethynyl phosphonic acid and another rinse to reduce nonspecific
adsorption as shown in (c), and finally hybridization with a complementary
oligonucleotide while still resisting nonspecific adsorption as shown in (d).
Experimental
Reagents
11-azidoundecyltrimethoxysilane was synthesized as previously described in
Chapter 3. TTMA (1,1',1''-Tris(1H-1,2,3-triazol-4-yl-1-acetic acid ethyl ester)
trimethylamine) was synthesized following a published procedure. 37 Ethynyl
phosphonic acid was synthesized according to a published procedure. 38,39,40
Oligonucleotide Sources
The Cy3-labeled, alkyne and amine-terminated oligonucleotides consisted of
32 bases with the following sequence: 5‟ - TCC ACT TAT CCT TGC ATC CAT CCT
CTG CCC TG – 3‟. This sequence is designated RG2. Cy3 dyes were attached to the
3‟ end, and terminal alkynes and amines were attached to the 5‟ end. For simplicity,
oligonucleotides in this study are named Cy3-RG2-alkyne and Cy3-RG2-amine. The
Cy3-RG2-amine oligonucleotide was purchased from Integrated DNA Technologies
(IDT). The Cy3-RG2-alkyne oligonucleotide was synthesized and purified by HPLC
at Helicos BioSciences by coupling an alkyne-terminated NHS (N-hydroxy
succinimide) linker to the Cy3-RG2-amine oligonucleotide. Figure S1 in the
Supporting Information shows the linker on this oligonucleotide. The Cy5-labeled
105

complementary oligonucleotide consisted of 32 bases with the following sequence: 5‟
- CAG GGC AGA GGA TGG ATG CAA GGA TAA GTG GA – 3‟. The Cy5 dye
was attached to the 3‟ end. The noncomplementary oligonucleotide had a Cy5 dye
attached to the 5‟ end and consisted of the following sequence: 5'- ACT GCT ACT
GCT ACT GCT ACT GCT ACT GCC CAC AAA CCA AAA GCC CAG ACA CCC
GGA GTT TTT TTT – 3‟.
Surface Cleaning
„Ultraclean‟ glass surfaces 40 mm in diameter (Erie Scientific) and single-side
polished Si(100) wafers were cleaned in an oxygen plasma cleaner (GaLa Instrumente
Plasma Prep5) for 10 min prior to vapor deposition with an oxygen flow rate of 100
std. mL/min using 50 percent relative power at a pressure of 0.25 mbar. This cleaning
method resulted in hydrophilic surfaces with water contact angles approaching 0°.
Monolayer Vapor Deposition
Siloxane monolayers were vapor-deposited onto silicon oxide surfaces in o-
ring sealed, glass vacuum desiccators fitted with Teflon stopcocks (Jencons part
number 250-048) with internal volumes of about 600 mL. Neat
azidoundecyltrimethoxysilane (100 µL) was pipetted onto and absorbed into 42.5 mm
diameter Whatman filter paper in the bottom of the desiccator. Water was deliberately
added to the depositions as a reactant for the hydrolysis reaction by adding 3 Å
molecular sieves (Acros) with adsorbed water into the bottom of the desiccator in a
foil boat. Water was adsorbed into the sieves by submerging 1.5 g of the as-received
sieves in water for 5 min., filtering the excess water using a fritted glass filter, and
then pumping on the sieves at room temperature for 5 minutes with a liquid nitrogen-
106

trapped mechanical pump. The plasma-cleaned surfaces were placed on a metal rack
in the chamber above the filter paper with absorbed silane. The desiccator was then
evacuated through a rubber hose to a glass vacuum line with a liquid nitrogen-trapped
mechanical pump for approximately 60 seconds. The final pressure at the trap was 1
Torr. The Teflon valve on the desiccator was closed, and the chamber was placed in a
110°C preheated oven for 12 h. After deposition, the valve was opened to ambient air
to return the chamber to atmospheric pressure and to remove the samples.
Surface Characterization
Bulk fluorescence emission spectra were collected from the glass surfaces
using a Horiba Jobin Yvon Fluorolog-3 spectrofluorometer equipped with a 450 watt
Xenon lamp. Cy3 emission spectra were collected from 545-700 nm using 530 nm
wavelength excitation light after passing through a bandpass filter and reaching the
glass surface at normal incidence. Emission light was collected using front face
detection and after passing through a bandpass filter. The bandpass on the emission
and excitation monochromators was 5 nm for all experiments. Data were collected
using an integration time of 0.5 s at 1 nm increments. Cy5 emission spectra were
collected under the same conditions except 640 nm excitation light and bandpass
filters for the Cy5 dye were used.
Single molecule fluorescence images were obtained using a custom-built
instrument at Helicos Biosciences 33 using 532 nm light for Cy3 excitation and 647 nm
light for Cy5 excitation.
107

Cu-Catalyzed Azide-Alkyne Cycloaddition
CuAAC reactions were performed by exposing azide-terminated monolayers to
aqueous solutions containing 10 nM of an alkyne-terminated oligonucleotide or
fluorescent dye, 500 µM CuSO4·5H2O, 500 µM TTMA, and 5 mM sodium ascorbate
in 3:2 dimethyl sulfoxide (DMSO):water for 1 h at room temperature. Next, the
surfaces were rinsedaccording to the standard buffer rinsing procedure outlined below.
Ethynyl phosphonic acid CuAAC conditions were as follows: 1 mM ethynyl
phosphonic acid, 500 µM CuSO4·5H2O, 500 µM TTMA, 5 mM sodium ascorbate in
3:2 DMSO:water solvent, for 15 minutes at room temperature. Conditions for the no
Cu(I) control experiments were the same except the CuSO4·5H2O was not added to the
reaction mixture.
Detergent and Buffer Rinsing Procedure
After exposure to oligonucleotides, the azide-terminated monolayers were
subjected to a standard rinse procedure: water, 1 time in 3X sodium saline citrate
(SSC), 2 times in 150 mM HEPES / 0.1% sodium dodecyl sulfate (SDS) / 1X SSC, 2
times in 150 mM HEPES/NaCl, and water. The solutions were heated to 37°C while
the water rinses were at ambient laboratory temperature. Approximately 100 mL of the
solutions were used for each rinse. This rinsing procedure will be called the standard
rinse in the rest of the paper.
Hybridization
Oligonucleotide hybridization experiments were performed by exposing
surfaces to 10 nM oligonucleotide solutions in 3X SSC at 37°C for 15 min. After
108

hybridization, the surfaces were rinsed 1 time in 3X SSC, 2 times in 150 mM HEPES/
0.1% SDS/ 1X SSC, and 2 times in 150 mM HEPES/NaCl.
Results and Discussion
Nonspecific Adsorption of Oligonucleotides onto Azide-Terminated Monolayers
Distinguishing between covalently attached and nonspecifically adsorbed
biomolecules is a common problem when verifying their attachment to surfaces. To
assess the nonspecific adsorption of dye-labeled oligonucleotides onto azide-
terminated monolayers, we exposed the monolayers to unreactive Cy3-labeled, amine-
terminated oligonucleotides (Cy3-RG2-amine). Figure 2a shows fluorescence
emission spectra of azide-terminated monolayers on glass before and after a 1 h
exposure to 10 nM Cy3-RG2-amine in water. After exposure, an increase in counts
above the background spectrum is observed even though the surfaces were rinsed with
detergent and buffer solutions according to the standard rinsing procedure. This
increase in fluorescence is attributed to Cy3 emission from nonspecifically adsorbed
Cy3-labeled oligonucleotides because it is observed at the same wavelengths observed
in the emission spectra of the Cy3-RG2-amine oligonucleotide in solution and for a
Cy3-alkyne dye covalently attached to a glass surface (Figures S2a and b in the
Supporting Information). In addition, the azide groups in the monolayer are unreactive
with the functional groups present on the Cy3-RG2-amine oligonucleotides.
Ethynyl Phosphonic Acid as a Blocking Agent to Reduce Nonspecific Adsorption
We hypothesized that the introduction of negative charge on the surface might
reduce the nonspecific adsorption of oligonucleotides by making the surface more
hydrophilic and by electrostatically repelling the negatively charged phosphate
109

ackbones of the oligonucleotides. Ethynyl phosphonic acid was synthesized and then
covalently attached to azide-terminated monolayers using the CuAAC reaction after
exposure to Cy3-labeled oligonucleotides. As shown in Figure 2b, no increase in
fluorescence above the background for the vapor-deposited azide-terminated
monolayer on glass was observed after a 1 h exposure of the monolayer to 10 nM
Cy3-RG2-amine in water, standard rinse, 15 min CuAAC reaction with ethynyl
phosphonic acid, and then another standard rinse. From this experiment, we infer that
the introduction of the ethynyl phosphonic acids onto the surface was successful in
reducing the nonspecific adsorption of Cy3-labeled oligonucleotides to the level of
noise in bulk fluorescence spectra.
XPS was used to confirm the covalent attachment of the ethynyl phosphonic
acid to an azide-terminated monolayer (see Figure S4 in the Supporting Information).
Commercially available propiolic acid was also successful as a blocking agent to react
with azide groups in the monolayer and to resist the nonspecific adsorption of
oligonucleotides as shown in Figure S5 in the Supporting Information.
110

counts per second
counts per second
1.0x10 3
7.5x10 2
5.0x10 2
2.5x10 2
0.0
1.0x10 3 (b)
7.5x10 2
5.0x10 2
2.5x10 2
0.0
(a)
Background
After Cy3-RG2-amine Exposure
Background
After Cy3-RG2-amine Exposure
and Ethynyl Phosphonic Acid
CuAAC Reaction
550 600 650 700
Wavelength / nm
Figure 2. Cy3 fluorescence emission spectra of vapor-deposited azide-terminated
monolayers on glass excited at 530 nm. (a) Emission of azide-terminated monolayer
before (dots) and after (solid) exposure to 10 nM Cy3-RG2-amine and standard rinsing.
(b) Emission of azide-terminated monolayer before (dots) and after (solid) exposure to
10 nM Cy3-RG2-amine followed by a standard rinse, 15 min ethynyl phosphonic acid
CuAAC reaction, and another standard rinse.
Covalent Attachment of Alkyne-Terminated Oligonucleotides to Monolayers
Once a method for reducing the nonspecific adsorption of Cy3-labeled
oligonucleotides onto azide-terminated monolayers was developed, the azide groups
111

were reacted with alkyne-terminated, Cy3-labeled oligonucleotides (Cy3-RG2-alkyne)
using CuAAC reaction conditions. Figure 3a shows emission spectra of an azide-
terminated monolayer on glass before and after a 1 h CuAAC reaction with 10 nM
Cy3-RG2-alkyne in water followed by a standard rinse, a 15 min CuAAC reaction
with ethynyl phosphonic acid, and another standard rinse. A peak is observed in the
fluorescence spectrum after the CuAAC reactions and rinsing, and it is assigned to
Cy3 emission from covalently attached Cy3-labeled oligonucleotides. To further test
against nonspecific adsorption, emission spectra were collected from an azide-
terminated monolayer before and after a 1 h exposure of an azide-terminated
monolayer to the same CuAAC reaction and rinsing conditions used in Figure 3a but
without the Cu(I) catalyst. Figure 3b shows that no increase in fluorescence above the
background is observed after exposure without the Cu(I) catalyst providing further
confirmation that the increase in fluorescence with Cu(I) present is attributable to
covalently attached Cy3-labeled oligonucleotides.
In addition to bulk fluorescence spectroscopy, single molecule fluorescence
spectroscopy was used to characterize the azide-terminated monolayers on glass after
exposure to Cy3-labeled oligonucleotides. Figure 3c shows a single molecule
fluorescence image after a 1 h CuAAC reaction with 0.1 nM Cy3-RG2-alkyne
followed by a standard rinse, 15 min ethynyl phosphonic acid CuAAC reaction, and
another standard rinse. The white objects in the image are attributable to fluorescence
emission on the surface resulting from the 532 nm excitation light. Quantification of
the objects results in approximately 1589 fluorescent objects per 1000 µm 2 . A single
molecule fluorescence image after a 1 h exposure of an azide-terminated monolayer to
112

0.1 nM Cy3-RG2-alkyne under the same conditions but without the Cu(I) catalyst is
shown in Figure 3d. The number density of fluorescent objects is 6 times less without
the Cu(I) catalyst present than with it, approximately 250 objects per 1000 µm 2 , which
is similar to the background number density of fluorescent objects measured on glass
surfaces after monolayer vapor deposition. The larger number density of fluorescent
objects in the single molecule image after exposure to the Cy3-labeled
oligonucleotides with the Cu(I) catalyst present is consistent with the bulk
fluorescence experiments and provides additional evidence that the alkyne-terminated
oligonucleotides are being covalently attached. The oligonucleotide concentration
used during the CuAAC reaction was decreased 100-fold from the 10 nM used for
bulk fluorescence measurements to 0.1 nM for the single molecule measurements to
control the number density of attached oligonucleotides so that single molecules could
be resolved. This experiment also demonstrates the utility of the vapor-deposited,
azide-terminated monolayers for single molecule fluorescence measurements because
their background fluorescence is low enough to clearly observe fluorescence emission
from dyes on the surface.
113

counts per second
counts per second
2000
1500
1000
500
0
2000
1500
1000
500
0
(a)
(b)
Background
After Cy3-RG2-alkyne CuAAC
Background
After Cy3-RG2-alkyne Exposure
550 600 650 700
Wavelength / nm
114
(c)
0.1 nM, with Cu(I), 1589 objects/1000 µm 2
(d)
0.1 nM, no Cu(I), 256 objects/1000 µm 2
Figure 3. Cy3 bulk fluorescence emission spectra and single molecule fluorescence
images of vapor-deposited azide-terminated monolayers on glass. (a) Emission of an
azide-terminated monolayer before (dots) and after (solid) a 1 h CuAAC reaction with
10 nM Cy3-RG2-alkyne. (b) Emission of an azide-terminated monolayer before (dots)
and after (solid) a 1 h exposure to 10 nM Cy3-RG2-alkyne without the Cu(I) catalyst.
(c) Single molecule fluorescence image of an azide-terminated monolayer after a 1 h
CuAAC reaction with 0.1 nM Cy3-RG2-alkyne. (d) Single molecule fluorescence
image of an azide-terminated monolayer after a 1 h exposure to 0.1 nM Cy3-RG2-
alkyne without the Cu(I) catalyst. Each CuAAC reaction and exposure was followed

y a standard rinse, a 15 min ethynyl phosphonic acid CuAAC reaction, and another
standard rinse. The bulk fluorescence spectra were collected using 530 nm excitation
light while 532 nm excitation light was used for the single molecule fluorescence
images.
Hybridization of Complementary Oligonucleotides to Covalently Attached
Oligonucleotides
To assess the viability of the single-stranded oligonucleotides after covalent
attachment to azide-terminated monolayers using the CuAAC reaction, hybridization
experiments with a Cy5-labeled complementary oligonucleotide were conducted.
Figure 4a shows Cy5 bulk fluorescence emission spectra of an azide-terminated
monolayer on glass that had been reacted with the Cy3-RG2-oligonucleotide and
ethynyl phosphonic acid before and after a 15 min exposure to 10 nM of a Cy5-labeled
complementary oligonucleotide in 3x SSC followed by rinsing. After exposure, an
increase in fluorescence above the background is observed that is consistent with Cy5
emission. 41 From the increase in fluorescence, we infer that Cy5-labeled,
complementary oligonucleotides hybridized to the covalently attached
oligonucleotides on the surface.
Two control experiments were performed to confirm the specific hybridization
of the Cy5-labeled complementary oligonucleotide on the surface. For the first control,
a nominally identical glass surface to the one used above was prepared by covalently
attaching the Cy3-RG2-alkyne and reacting the remaining azide groups with ethynyl
phosphonic acid. This surface was then exposed to 10 nM of a non-complementary,
115

Cy5-labeled oligonucleotide using the same hybridization conditions as used for the
complementary oligonucleotide. As shown in Figure 4b, no increase in fluorescence
was observed after exposure and rinsing. This result suggests that the non-
complementary oligonucleotide did not hybridize as expected and that it did not
nonspecifically adsorb onto the surface as measured using bulk fluorescence.
A second control experiment was performed that tested the nonspecific
adsorption of the Cy5-labeled complementary oligonucleotide onto an azide-
terminated monolayer that had been reacted with ethynyl phosphonic acid. Figure 4c
shows that the Cy5 fluorescence emission spectra are the same before and after
exposure of the phosphonic acid-terminated monolayer to 10 nM of the Cy5-labeled
complementary oligonucleotide followed by standard rinsing. It is inferred that the
Cy5-labeled oligonucleotide did not nonspecifically adsorb onto the surface because
no increase in fluorescence was observed after exposure. Overall, the experiments
represented in Figure 4 demonstrate the utility of azide-terminated siloxane
monolayers on glass for the covalent attachment and subsequent hybridization of
oligonucleotides while resisting nonspecific adsorption.
116

counts per second
counts per second
counts per second
2000
1500
1000
500
0
2000
1500
1000
500
0
2000
1500
1000
500
0
(a)
(b)
(c)
650 700 750 800
Wavelength / nm
117
Background
After Hybridization
Background
After Exposure
Background
After Exposure
Figure 4. Cy5 fluorescence emission spectra of azide-terminated monolayers on glass
exposed to hybridization conditions for Cy5-labeled oligonucleotides. (a) Azide-
terminated monolayer reacted for 1 h with 10 nM Cy3-RG2-alkyne before and after

exposure to the Cy5-labeled complementary oligonucleotide and standard rinsing. (b)
Azide-terminated monolayer reacted for 1 h with 10 nM Cy3-RG2-alkyne before and
after exposure to a Cy5-labeled non-complementary oligonucleotide and standard
rinsing. (c) Azide-terminated monolayer reacted with ethynyl phosphonic acid before
and after exposure to the Cy5-labeled complementary oligonucleotide and standard
rinsing. 640 nm excitation light was used for all spectra.
Resistance to Nonspecific Adsorption during Single Molecule Sequencing by
Synthesis
The strategy developed in this paper for resisting the nonspecific adsorption of
dye-labeled oligonucleotides was applied to resisting the nonspecific adsorption of
dye-labeled mononucleotides during single molecule sequencing by synthesis. A
major challenge for sequencing by synthesis technologies is the development of
surfaces that resist the nonspecific adsorption of oligo- and mono-nucleotides to
achieve predictable rinsing on the surface. The inability to rinse unincorporated
mononucleotides from the surface can lead to increased substitution and error rates
during sequencing. 2
To test our strategy, azide-terminated monolayers were first reacted with an
alkyne-terminated oligonucleotide followed by a second reaction of the azides with
propargyl alcohol as a blocking agent. Next, oligonucleotides templates were captured
on the surface via hybridization to covalently attached oligonucleotides. Using the
single molecule sequencing by synthesis platform developed by Helicos
BioSciences, 33 the surfaces were then sequentially exposed to dye-labeled
mononucleotides in the presence of DNA polymerase for the template-driven
118

enzymatic extension at the 3‟-end of the covalently attached oligonucleotide strands.
The surfaces were then rinsed according to the same aqueous and buffer rinses used in
the oligonucleotide hybridization experiments. Single molecule fluorescence
spectroscopy was used to confirm the template-driven enzymatic addition of the dye-
labeled mononucleotides to the surfaces and to quantify unincorporated dye-labeled
mononucleotides that were not removed by rinsing.
Table S1 in the Supporting Information shows the number of uncorrelated
fluorescent objects measured during single molecule sequencing on azide-terminated
monolayers that were reacted with alkyne-terminated oligonucleotides and propargyl
alcohol. The uncorrelated objects are attributed to unincorporated, nonspecifically
adsorbed dye-labeled mononucleotides that remained on the surface after rinsing. For
comparison, Table S2 shows the number of unincorporated mononucleotides for
commercially produced epoxide surfaces that were reacted with amine-terminated
oligonucleotides and phosphate. The number of unincorporated mononucleotides
above the background fluorescence is approximately 4-5 times lower on the azide-
terminated monolayers than for the epoxide-terminated monolayers. Perhaps there is a
higher density of hydroxyl groups, which have been shown to resist the nonspecific
adsorption of nucleotides, 42 on the azide-terminated monolayers after reaction with
propargyl alcohol than on the epoxide surfaces after reaction with phosphate. 43 The
longer, 11-carbon alkyl chains in the azide-terminated monolayers should also
promote denser, more uniform monolayers compared to the shorter, 3-carbon alkyl
chains in the epoxide-terminated monolayers. The increase in monolayer density and
uniformity may be important for resisting the nonspecific adsorption of nucleotides.
119

Conclusions
In summary, we have demonstrated a strategy for covalently attaching alkyne-
terminated oligonucleotides onto azide-terminated monolayers on glass surfaces that
were viable for hybridization with complementary strands in solution with minimal
nonspecific adsorption. CuAAC reactions between azide groups and ethynyl
phosphonic acid reduced the nonspecific adsorption of dye-labeled oligonucleotides to
the level of noise in bulk fluorescence spectra. An optimized procedure involved an
initial CuAAC reaction to covalently attach alkyne-terminated oligonucleotides
followed by a second CuAAC reaction with ethynyl phosphonic acid to create surfaces
that resist nonspecific adsorption but allow complementary oligonucleotide targets to
be captured via hybridization. Bulk and single molecule fluorescence measurements
were used to confirm the presence of dye-labeled oligonucleotides on the surfaces.
Acknowledgments
This work was supported by funding from Helicos BioSciences Corporation. The
authors acknowledge NSF grant CHE-0639053 for support of the spectrofluorometer.
The authors also acknowledge NSF grant DMR-0213618 to the Center on Polymer
Interfaces and Macromolecular Assemblies for use of the plasma cleaner.
120

Supporting Information
Figure S1. Reaction scheme for the synthesis of the alkyne-terminated oligonucleotide
that was used in this study for CuAAC reactions with azides. The synthesis was
performed by reacting an NHS-activated alkyne with the primary amine on the 5‟ end
of a Cy3-labeled oligonucleotide as shown. .
Cy3 Fluorescence in Solution and on Glass Surfaces
Figure S2a shows a fluorescence emission spectrum of an aqueous solution
containing 10 nM of the Cy3-labeled oligonucleotide, Cy3-RG2-amine, excited at 530
nm. A peak is observable in the emission spectrum at 565 nm, consistent with Cy3
fluorescence. 41 A Cy3 dye with a terminal alkyne (Cy3-alkyne) was synthesized to test
the detection of Cy3 fluorescence on glass surfaces. Its chemical structure is shown in
Figure S3. The CuAAC reaction was used to covalently attach the Cy3-alkyne to
azide-terminated siloxane monolayers. Figure S2b shows fluorescence emission
spectra of an azide-terminated monolayer on glass before and after a 1 h CuAAC
121

eaction performed by exposing the surface to 10 nM of the Cy3-alkyne in water with
the Cu(I) catalyst present followed by a standard rinse, CuAAC reaction with ethynyl
phosphonic acid, and another standard rinse. After the reaction, an increase in
fluorescence above the background spectrum is observed in the same wavelength
region as observed for the Cy3-labeled oligonucleotide in solution. This increase in
fluorescence is attributed to emission from covalently attached Cy3 dye molecules.
Figure S2c shows fluorescence emission spectra of an azide-terminated monolayer
before and after a 1 h exposure to 10 nM of the Cy3-alkyne without the Cu(I) catalyst
followed by the standard rinsing procedure. No increase in fluorescence is observed
without the Cu(I) catalyst providing further evidence that the Cy3-alkyne is covalently
attached to the surface when the Cu(I) catalyst is present and that fluorescence
spectroscopy can be used to detect the presence of Cy3 dyes on glass surfaces.
122

counts per second
counts per second
counts per second
3.0x10 5
2.5x10 5
2.0x10 5
1.5x10 5
1.0x10 5
5.0x10 4
0.0
3.0x10 3
2.5x10 3
2.0x10 3
1.5x10 3
1.0x10 3
5.0x10 2
0.0
3.0x10 3
2.5x10 3
2.0x10 3
1.5x10 3
1.0x10 3
5.0x10 2
0.0
(a)
(b)
(c)
550 600 650 700
Wavelength / nm
123
10 nM Cy3-RG2-amine
in water
Background
After Cy3-alkyne CuAAC
Background
After Cy3-alkyne exposure
Figure S2. (a) Cy3 fluorescence emission spectrum of 10 nM of a Cy3-labeled
oligonucleotide, Cy3-RG2-amine, in water excited at 530 nm. Cy3 fluorescence
emission spectra of vapor-deposited azide-terminated monolayers on glass before
(dots) and after (solid) exposure to 10 nM Cy3-alkyne with (b) and without (c) the

Cu(I) catalyst for the CuAAC reaction present. The glass surfaces were excited with
530 nm light. After exposure and reaction, the surfaces were subjected to: a standard
rinse, a CuAAC reaction with ethynyl phosphonic acid, and another standard rinse.
Figure S3. Chemical structure of the alkyne-terminated Cy3 dye (Cy3-alkyne) that
was covalently attached to azide-terminated monolayers using the CuAAC reaction.
XPS Confirmation of Reaction with Ethynyl Phosphonic Acid
X-ray photoelectron spectroscopy (XPS) provides a convenient probe for
monitoring CuAAC reactions on azide-terminated monolayers, 17,44 and it was used to
confirm the covalent attachment of ethynyl phosphonic acid. Figure S4 shows a high-
resolution N 1s spectrum of an azide-terminated monolayer on silicon oxide. An
asymmetric doublet is present because the azide group contains nitrogen atoms in two
chemically distinct environments. The higher binding energy peak is attributed to the
electron-deficient, central nitrogen atom while the lower binding energy peak is
assigned to the outer, more electron-rich nitrogen atoms. 45 Therefore, the peak ratio
should ideally be 1:2 as shown here. After a CuAAC reaction with ethynyl phosphonic
124

acid, the higher binding energy peak disappears leaving only one N 1s peak present in
the high-resolution XPS spectrum (Figure S4d), which is attributable to the three
nitrogen atoms in the triazole product. The quantitative reaction of the azide groups is
inferred from the disappearance of the higher binding energy N 1s photoelectron peak.
In addition, a new peak is observed in the high-resolution P 2p spectrum at 133 eV
after the CuAAC reaction with ethynyl phosphonic acid; whereas, no P 2p was present
before reaction as shown in Figures S4a and b. The presence of the P 2p peak after the
reaction is attributed to the phosphorus atoms in the covalently attached ethynyl
phosphonic acid molecules. 46
Counts
Counts
(a)
100
(b)
100
P 2p before
CuAAC
P 2p after
CuAAC
125 130 135 140 390 400 410
Binding Energy / eV
125
(c)
500
(d)
500
N 1s before
CuAAC
Binding Energy / eV
N 1s after
CuAAC
Figure S4. High-resolution XPS spectra of the P 2p [(a) and (b)] and N 1s [(c) and
(d)] photoelectron regions for an azide-terminated monolayer on silicon oxide before
and after a CuAAC reaction with 10 mM ethynyl phosphonic acid in 3:2 DMSO:H2O.
Counts
Counts

Spectra were obtained using a Surface Science Model 150 spectrometer with Al Kα
radiation (1486.6 eV). 10 scans were collected at 0.1 eV resolution at a takeoff angle
of 35° from the surface.
Propiolic Acid as a Blocking Agent to Reduce Nonspecific Adsorption
Figure S5 shows fluorescence emission spectra for azide-terminated
monolayers on glass exposed to 10 nM of the alkyne-terminated oligonucleotide, Cy3-
RG2-alkyne. Figure S5a shows an increase in fluorescence above the background
when an azide-terminated monolayer was exposed to 10 nM of the oligonucleotide for
1 hour without Cu(I) present followed by standard rinsing without propiolic acid.
Figure S5b shows that reacting propiolic acid with the azide groups in the monolayer
after exposure to the Cy3-RG2-alkyne and standard rinsing reduces the nonspecific
adsorption of oligonucleotides to the level of the background. Figure S5c shows
fluorescence emission spectra before and after CuAAC reaction with 10 nM of the
Cy3-RG2-alkyne followed by a standard rinse, CuAAC reaction with propiolic acid,
and another standard rinse. As in the Cy3-alkyne studies, addition of the Cu(I) catalyst
results in covalent attachment of the alkyne-terminated oligonucleotide as inferred
from the increase in the fluorescence emission above the background.
126

counts per second
counts per second
counts per second
1000
750
500
250
0
1000
750
500
250
0
2000
1500
1000
500
0
(a)
(b)
(c)
550 600 650 700
Wavelength / nm
127
Background
After Exposure
Background
After Exposure
Background
After CuAAC
Figure S5. Fluorescence emission spectra of azide-terminated monolayers on glass
before and after a 1 h exposure to 10 nM of the Cy3-RG2-alkyne: (a) without Cu(I)
present and standard rinsing; (b) without Cu(I) present followed by a standard rinse,

propiolic acid CuAAC, and another standard rinse; (c) with Cu(I) present followed by
a standard rinse, propiolic acid CuAAC, and another standard rinse.
Monolayer Resistance to Nonspecific Adsorption During Single Molecule
Sequencing by Synthesis
Once the nonspecific adsorption of dye-labeled oligonucleotides was reduced
to the level of noise as measured by bulk fluorescence and single molecule
fluorescence (Figure 3), the nonspecific adsorption of dye-labeled mononucleotides
onto the monolayers was characterized using single molecule fluorescence
spectroscopy, a more sensitive probe. Table S1 shows the average number of
uncorrelated fluorescent objects per 1000 µm 2 for 180 measurements during single
molecule sequencing by synthesis on vapor-deposited, azide-terminated monolayers
that were reacted with alkyne-terminated oligonucleotides and commercially available
propargyl alcohol as a blocking agent followed by a standard rinse. The uncorrelated
objects represent nonspecifically adsorbed, dye-labeled nucleotides on the surface that
were not incorporated into a growing primer-template strand. It is inferred that the
surfaces exhibit high resistance to the nonspecific adsorption of dye-labeled
nucleotides because the number of uncorrelated objects is only a few tens of counts
above the background.
128

Table S1. Nonspecific Adsorption of Dye-Labeled Mononucleotides onto Azide-
Terminated Monolayers Reacted with Alkyne-Terminated Oligonucleotides and
Propargyl Alcohol during Single Molecule Sequencing by Synthesis
Average rinse for each nucleotide base (objects per 1000 µm 2 )
Background (X) Adenine (A) Cytosine (C) Guanine (G) Uracil (U)
145.5 188.6 168.3 169 190.2
Average rinse minus background (objects per 1000 µm 2 )
A-X C-X G-X U-X
43.1 22.8 23.5 44.8
Number of positions (cycles) sampled for calculation
390 180 180 180 180
For comparison, a similar experiment was performed on epoxide-terminated
siloxane monolayers that were commercially vapor deposited onto glass surfaces and
then reacted with amine-terminated oligonucleotides followed by a standard rinse and
blocking in a phosphate solution. 43 Table S2 shows the average number of
uncorrelated fluorescent objects per 1000 µm 2 for 240 measurements on these surfaces
during single molecule sequencing by synthesis. The azide-terminated monolayers
reacted with propargyl alcohol are more resistant to the nonspecific adsorption of dye-
labeled mononucleotides because the numbers of uncorrelated objects minus the
129

ackgrounds for each base are lower than the number of uncorrelated objects
measured on the epoxide-terminated monolayers.
Table S2. Nonspecific Adsorption of Dye-Labeled Mononucleotides onto Epoxide-
Terminated Monolayers Reacted with Amine-Terminated Oligonucleotides and
Phosphate during Single Molecule Sequencing by Synthesis
Average rinse for each nucleotide base (objects per 1000 µm 2 )
Background (X) Adenine (A) Cytosine (C) Guanine (G) Uracil (U)
50.2 246.6 198.9 147.5 288.4
Average rinse minus background (objects per 1000 µm 2 )
A-X C-X G-X U-X
196.4 148.7 97.3 238.2
Number of positions (cycles) sampled for calculation
510 240 240 240 240
130

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