NOVEL METHODS FOR INFRARED SPECTROELECTROCHEMICAL CHARACTERIZATION OF HYDROGENASE FILMS

by Joshua A. Johnson © Copyright by Joshua A. Johnson, 2014

All Rights Reserved A thesis submitted to the Faculty and the Board of Trustees of the Colorado School of Mines in partial fulfillment of the requirements for the degree of Master of Science (Applied Physics).

Golden, Colorado Date

Signed: Joshua A. Johnson

Signed: Dr. Thomas E. Furtak Thesis Advisor

Golden, Colorado Date

Signed: Dr. Thomas E. Furtak Professor and Department Head Department of Physics

ii ABSTRACT

Novel methods and materials including nanoporous gold leaf films, attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy with optically coupled wafers and biocompatible gold coated doped silicon electrodes were developed for the spec- troelectrochemical study of anaerobic enzymes capable of catalyzing the reversible oxidation

and reduction of hydrogen known as hydrogenase. The study of hydrogenases themselves and their interaction with inorganic substrates is essential for the future development of biohybrid, biomimetic or bio-inspired devices capable of the conversion of solar energy to hydrogen fuel. In this work, [FeFe]-hydrogenase I from Clostridium acetobutylicum (CaI) is studied to understand the mechanism by which enzymes activate hydrogen using base metals as opposed to noble metals. Due to the limitation of the quantity of available hy- drogenase and the nature of spectroelectrochemistry, novel methods presented in this work focus on optimizing the efficiency with which proteins are used in addition to maximizing IR absorbance and electrochemical stability.

Confinement of proteins using a nanoporous gold leaf film was shown to maximize the concentration of proteins near the reflection plane of an attenuated total reflectance (ATR) prism while still allowing proteins to interact with a bulk electrolyte. Optically coupling wafers to an ATR prism allowed for the rapid testing of organic functionalizations or for other treatments deemed too extreme for immediate testing with a delicate and expensive ATR crystal. Biocompatible gold coated doped silicon electrodes were designed as a method of efficiently transferring charge to an adsorbed protein while remaining transparent in the infrared. The drying of CaI films for the purpose of concentrating as much as possible on an

ATR prism surface revealed IR spectra as of yet to be seen in the literature. Novel techniques were found to be useful for general spectroelectrochemistry and study of other proteins as was demonstrated with various redox couples and the common protein, myoglobin.

iii TABLE OF CONTENTS

ABSTRACT ...... iii

LISTOFFIGURES ...... vi

LISTOFTABLES ...... ix

LISTOFABBREVIATIONS ...... x

ACKNOWLEDGMENTS ...... xii

CHAPTER1 INTRODUCTION ...... 1

1.1 Hydrogenases: generalproperties ...... 4

1.2 Hydrogenases: uses and challenges in devices ...... 6

1.3 Objectives...... 9

1.4 Previouswork...... 10

CHAPTER 2 METHODS AND MATERIALS ...... 15

2.1 Transmission cells ...... 15

2.2 Attenuatedtotalreflectionsetup...... 16

2.3 Flowcells ...... 18

2.4 Optically Coupled wafers ...... 19

2.5 Electrochemicalsetup...... 21

2.6 Rapid electrochemical modulation ...... 21

2.7 Functionalizations of Silicon ...... 23

2.8 PreparationofProteins...... 24

2.9 Working electrode fabrication ...... 26

iv 2.9.1 Semi-conductor electrodes: Doped silicon ...... 26

2.9.2 Evaporatedgoldthin-films ...... 28

2.9.3 Nanoporousgoldleaf ...... 29

CHAPTER 3 RESULTS AND DISCUSSION ...... 31

3.1 ScopeandConstraintsofThisStudy ...... 31

3.2 Optically Coupled Wafers ...... 32

3.3 DopedSielectrode ...... 34

3.4 Au-coateddopedSielectrodes ...... 37

3.5 NPGLfilms ...... 41

3.6 Confinementofmyoglobinfilms ...... 43

3.7 AffectsofdryingonCaIfilms ...... 46

CHAPTER4 CONCLUSION...... 50

REFERENCESCITED ...... 52

APPENDIX - ALTERNATIVE METHODS AND MATERIALS EXPLORED . . . . . 57

A.1 CO inhibition of Myoglobin ...... 57

A.2 ElectrolessGold...... 59

A.2.1 Smallareadepositions ...... 60

A.2.2 Largeareadepositions ...... 61

A.3 EvaporatedAu ...... 65

A.4 Doped Si fabrication and characterization details ...... 68

A.5 Carboxyethylsilanetriol Monolayer Fabrication and Characterization...... 68

A.6 Meltmount...... 75

A.7 NPGLenhancementstudies ...... 75

v LIST OF FIGURES

Figure 1.1 Depiction of an ideal solar hydrogen economy ...... 3

Figure 1.2 CpIStructure...... 5

Figure1.3 H-clusterstructure ...... 6

Figure 1.4 Calculated catalytic intermediate steps for [FeFe]-hydrogenases...... 7

Figure 1.5 Hypothetical intermediates state of the H-cluster ...... 8

Figure 1.6 Covalently immobilized Hydrogenase ...... 9

Figure 1.7 Oxidized spectrum of CaI ...... 11

Figure 1.8 CO-inhibited spectrum of CaI...... 12

Figure 1.9 Reduced spectrum of CaI ...... 13

Figure 1.10 CO-inhibited spectrum of CaIusingATR ...... 13

Figure 2.1 Passage of IR beam through a contained sample volume ...... 16

Figure 2.2 Limitations of angle of incidence (θi) and how angle affects both the number of reflections and the penetration depth...... 18

Figure 2.3 Dimensions of flow cell ...... 19

Figure 2.4 Silicon wafer with ideal optical coupling to ZnSe ATR ...... 20

Figure2.5 StructureofMyoglobin...... 25

Figure 2.6 N-type Semiconductor-Electrolyte Interface at Equilibrium...... 27

Figure 2.7 SEM image of NPGL etched for 35 minutes in concentrated nitric acid courtesyofPeterCiesielski...... 30

Figure 3.1 Optically coupled wafers with spacers ...... 33

Figure 3.2 Optically coupled wafers without spacers ...... 34

vi Figure 3.3 Difference spectra of diiodomethane coupling fluid ...... 35

Figure 3.4 Cyclic Voltammograms of 10 mM Methyl Viologen in Tris buffer sweeping from −1.4V to 0V at 10mV/s...... 36

Figure 3.5 10 mM Methyl Viologen in Tris buffer oxidized at −1100 mV and reduced at −100 mV in succession with doped Si ATR...... 37

Figure 3.6 Spectroelectrochemical characterization of APTES functionalized, dopedSiwafersopticallycoupletoATRprism...... 38

Figure 3.7 Proof of working electrochemical system by demonstration of classic ferri/ferrocyanide cyclic voltammograms...... 39

Figure 3.8 Cartoon of incomplete Au coating with an MPA SAM and immobilized protein...... 39

Figure 3.9 (A) Decaying oxidized-minus-reduced spectra of ferri-/ferrocyanide using Au thin-film on a doped silicon wafer with numbers corresponding first, second, or third redox cycle. (B) Initial CV . .... 40

Figure 3.10 (A) Oxidized-minus-reduced spectra of ferri-/ferrocyanide using NPGL on ZnSe ATR. (B) Corresponding CV’s. For both graphics blue is initial data and yellow is final data...... 42

Figure 3.11 Cyclic voltammogram of methyl viologen using NPGL films as a workingelectrode ...... 43

Figure 3.12 Spectra of methyl viologen in an (a) oxidized state, (b) reduced, (c) oxidizedstateagain...... 44

Figure 3.13 Cartoon of myoglobin films confined by a NPGL membrane and chemical interactions with a bulk solution...... 45

Figure 3.14 Confined Mb films repeatedly CO inhibited. CO saturated solutions of NaDT in PBS were injected at time points labeled CO. PBS buffer equilibrated with ambient oxygen levels is injected at 1 mL/min starting at time points labeled O2. Time increments between collected spectra were ≈ 8 min. Any peaks other than the Fe-CO peak at 1967 cm−1 areduetowatervapor...... 46

Figure 3.15 Spectra of CaIinvariousstagesofdrying ...... 48

Figure 3.16 Comparison of CaI films dried (a) in a transmission cell, (b) on a horizontal ATR prism at NREL (c) on a vertical ATR prism at Mines . . 49

vii Figure A.1 Partially hydrated Mb film with composite spectrum in blue, hydrated film component in purple and dehydrated film component in yellow. . . . 57

Figure A.2 Double water bath for thermal isotropy during electroless Au deposition. . 62

Figure A.3 AFM images of electroless Au attempt ...... 63

FigureA.4 AFMimageof3minAu ...... 64

Figure A.5 Minicell setup ...... 66

Figure A.6 CV of 0.5 M H2SO4 with thick Au working electrode using the small volume flow cell (red), and the minicell (blue) ...... 66

Figure A.7 20 nm evaporated Au film on Si after rinsing with DI and acetone (left). Electrochemically cleaned 20 nmevaporated Au film (right) . . . . 67

Figure A.8 Cyclic voltammograms of ferricyanide using MPA functionalized Au coated doped Si using pH 4 PBS (left) and pH 7 PBS (right)...... 67

Figure A.9 Successive Etching of Highly Doped Si Wafers. Perturbations in spectra are due to variances in CO2andwatervapor...... 69

Figure A.10 Sessile water contact angle measurements for temperature dependent depositionofCSSmonolayers ...... 71

Figure A.11 Sessile water contact angles of acidic and basic drops on CSS monolayers deposited with different pH levels and corresponding thicknesses as determined by ellipsometry ...... 72

Figure A.12 AFM images with 512 samples at 1.001 Hz from Si wafer functionalized withCSSatvaryingpHlevels...... 73

Figure A.13 CSS monolayers formed on Si ATR surface and subsequent deprotonationwithNaOH...... 74

Figure A.14 Difference spectra between scans taken every ten minutes...... 76

Figure A.15 19.5 hr MPA deposition on NPGL film etched for various times: (a) 15min;(b)35min;(c)3hrs;(d)1day...... 77

Figure A.16 Surface area enhancement of NPGL ...... 78

viii LIST OF TABLES

Table 3.1 Comparison of best data with Mb in solution ...... 43

Table A.1 Comparison of methods with CO-Mb films with ZnSe prisms ...... 58

Table A.2 Comparison of methods with CO-Mb films with Si prisms ...... 58

ix LIST OF ABBREVIATIONS

Absorbanceunits...... A.U.

Aminopropyltriethoxylsilane ...... APTES

Atomicforcemicroscopy ...... AFM

Attenuatedtotalreflection...... ATR

Carbonmonoxide,carbonyl ...... CO

Carboxyethylsilanetriol salt ...... CSS

Centimeter ...... cm

ColoradoSchoolofMines ...... CSM

Cyanide,cyano ...... CN

di(thiomethyl)amine ...... DTMA

Ferricyanide...... Fe(CN)6

Fourier-transforminfrared ...... FTIR

Gram ...... g

Hydrogenase I from Clostridium acetobutylicum ...... CaI

Hydrogenase from Chlamydomonas reinhardtii ...... CrHydA1

Infrared ...... IR

Internalreflectionelement ...... IRE

Iron-Ironhydrogenase ...... [FeFe]-hydrogenase

Kilogram ...... kg

Nanoporousgoldleaf...... NPGL

x NationalRenewableEnergyLaboratory ...... NREL

3-Mercaptopropionicacid ...... MPA

Methylviologen...... MV

Millivolts ...... mV

Myoglobin...... Mb

Phosphatebufferedsaline ...... PBS

Polarization Modulation Infrared Reflection Adsorption Spectroscopy . . . . . PM-IRRAS

Self-assembledmonolayer ...... SAM

Silicon...... Si

Spectroelectrochemical ...... SEC

Surface-enhancedinfraredabsorption ...... SEIRA

Surface-enhanced infrared absorption spectroscopy ...... SEIRAS

Standardhydrogenelectrode...... SHE

SodiumDithionite ...... NaDT

Triethoxysilylbutyraldehyde ...... TESBA

Zincselenide ...... ZnSe

xi ACKNOWLEDGMENTS

I would like to thank Dr. Thomas Furtak, Dr. Reuben Collins and Dr. Paul King for their patience and support as I learned to be a better researcher. I would also like to thank Michael Ratzloff for his help in getting me started and help working in the labs at NREL. I would also like to gratefully acknowledge funding by the U.S. Department of Energy, Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences, for work on this project in addition to funding by the Trefny Institute for Educational Innovation.

xii CHAPTER 1 INTRODUCTION

The goal of this research is to provide a superior platform to study hydrogen activating metalloenzymes in biocompatible nanostructured materials for the purpose of creating a vi- able biohybrid device for renewable energies. The finite nature of humanity’s primary energy source, fossil fuels, provide a definite imperative towards finding renewable energy sources for future generations. When finding renewable energy sources the rate at which any source is replenished must be considered, which is to say that it must also be a sustainable source. Fossil fuels are technically renewable, however, new fossil fuel reserves require several million years to form [1]. Since humanity is consuming at a rate several orders of magnitude greater than reserves are created, fossil fuels are certainly not sustainable. In addition, burning of fossil fuels to produce energy releases carbon dioxide into the atmosphere which increasingly absorbs space-bound infrared radiation enhancing the greenhouse effect [2]. Thus the supply of ’clean’ sustainable energy is desired. The Sun is one source that, before vaporizing the planet, will provide Earth with clean, usable energy for the next 12 billion years [3]. Al- though this is sufficient for long term needs, the day-to-day energy requirements of citizens in a specific locality don’t match the availability of the Sun. Given that the total energy from the Sun that reaches the Earth in about an hour exceeds the total demand for energy of the world in a year then the ability to store even a fraction of this energy would be sufficient for powering civilization [4, 5]. Technically, fossil fuels are hydrocarbons with the energy from the Sun millions of years ago stored in their chemical bonds. If the concept of storing energy in chemical bonds can be applied to substances without carbon and on much faster time scales then the qualifications of a clean, sustainable energy source would be satisfied. The most simple and promising substance is dihydrogen gas (H2). The energy stored in the bond between hydrogen atoms in H2 has the highest energy density on a per mass basis of any

1 other substance (with the exception of nuclear fuels sources) at 142 MJ/kg[6]. Thus an en- ergy storage method of particular interest is in hydrogen electrolyzers which use electricity to convert hydrogen ions to hydrogen gas. In combination with solar cells, electrolyzers would serve to convert solar energy to chemical energy. When needed, the energy in hydrogen must be converted back into electricity. The combustion of hydrogen with oxygen will release the chemical energy as thermal energy with water as the only product and the thermal energy can then be converted to electricity. The intermediate conversion to thermal energy, how- ever, is inconvenient for practical considerations and thus extensive research has been done in hydrogen fuel cells which operate on the same principles as a hydrogen electrolyzer but in reverse. The combination of all these devices would form the basis for clean sustainable energy as shown in Figure 1.1; electricity from a solar cell is used in hydrolysis to cleave water into hydrogen and oxygen acting as energy reserves and, when needed, the energy stored in the bonds between hydrogen and oxygen is harvested in a fuel cell and converted back into water. In a canonical hydrolysis experiment, a sufficient voltage applied between two platinum or palladium electrodes oxidizes water into oxygen gas and hydrogen ions at the anode and reduces hydrogen ions into hydrogen gas at the cathode. The voltage applied is typically more than the thermodynamically determined potential for hydrolysis. The difference is known as the overpotential which is used as measure of efficiency. An electrochemical cell using noble metals such as platinum or palladium has a low overpotential however the low abundance of these elements on Earth make them economically infeasible for use in large- scale hydrogen production devices. Fortunately, nature has provided an efficient alternative in the form of enzymes called hydrogenases (H2ases). Enzymes are biological catalysts that facilitate reactions within living organisms and have been fine tuned through evolution to be efficient while still using common organic elements. The efficiency and elemental cost effectiveness of enzymes makes the basic study of how they work highly valuable given the potential impacts such knowledge would have on renewable energy. In fact, an overall goal

2 Figure 1.1: Depiction of an ideal solar hydrogen economy. Used with permission from the Royal Society of Chemistry. Copyright 2012.

3 of large scale research efforts in places such as the National Renewable Energy Laboratory (NREL) is to integrate nano and biological materials for solar energy conversion to fuels. Such a goal requires the massing of information about how and why certain proteins such as hydrogenase function so well in addition to information about how and why they function

differently when paired with inorganic materials. Not all the information about nano and biological materials is so easily obtained and for this reason novel methods must be explored to create a superior platforms for the study of proteins in biocompatible materials.

1.1 Hydrogenases: general properties

Hydrogenases are enzymes, found in the early 20th century, that are able to reversibly catalyze the oxidation of hydrogen gas into hydrogen ions by the following reaction [7]:

+ − 2H +2e ⇋ H2

Hydrogenase can further be classified as a metalloenzyme due to the composition of its reactive center. The two major subclasses of hydrogenases are nickel-iron ([NiFe]) hydro- genases and iron-iron ([FeFe]) hydrogenases. Another subclass of hydrogenase not typically

considered due to it’s limited capability of only partially cleaving hydrogen is single iron ([Fe]) hydrogenase. The focus of this body of work is on [FeFe]-hydrogenases and more specifically the [FeFe]-hydrogenase extracted from the bacteria Clostridium acetobutylicum and thus referred to as CaI. The ’I’ designation is to distinguish it from another hydrogenase expressed within Clostridium acetobutylicum which has a significantly different structure and

is preferential to the oxidation of hydrogen. CaI was chosen as the preferred hydrogenase to study the research team led by Paul King, an adviser and collaborator at the National Re- newable Energy Laboratory (NREL), because of the proteins equal capacity to both oxidize hydrogen gas and reduce hydrogen ions. The structure of Clostridial hydrogenases are all

similar in structure and catalytic activity [8]. The general structure, modeled in Figure 1.2, supports the active site, three [4Fe4S] accessory clusters, and a single [2Fe2S] cluster. The

4 Figure 1.2: (A) Modeled structure of Clostridium pasteurianum [FeFe]-hydrogenases with inorganic cofactors of CpI. Adapted with permission from Mulder et al. [9]. Copyright 2011 Elsevier. iron-sulfur clusters form an electron transport chain to and from the active site or so called H-cluster which is itself a [4Fe4S] subcluster covalently bonded to an [2Fe2S] subcluster by a bridging cysteine. The two irons are somewhat unusually ligated as both have a carbon monoxide (carbonyl- or CO) ligand and a cyanide (cyano- or CN) ligand. The proximal and distal irons are bridged by a di(thiomethyl)amine (DTMA) molecule. The structure of the protein surrounding the active site controls the reactivity of the H-cluster including substrate and product transfer. For example, it has been shown (Figure 1.3) that the Cysteine 298 (C298) residue in CaI is essential for proton transfer to the active site and thus necessary for any catalytic activity [10]. Although the exact process by which hydrogen gas is made within the active site of hy- drogenases is still under investigation, quantum mechanical and molecular mechanical simu- lations have been used to attempt to provide some insight [11]. The cycle shown in Figure 1.4 is a model derived from structural and biophysical data collected on Desulfovibrio desulfuri- cans hydrogenase which contains the same active site structure as other [FeFe]-hydrogenases including CaI [8]. The DTMA bridging the proximal and distal irons is thought to facilitate

5 Figure 1.3: C298 residue near [FeFe]-hydrogenase active site. Copied from Morra et al. [10]. Copyright 2012 PLoS ONE.

proton transfer near the active site. Experimental studies with [FeFe] hydrogenases from Chlamydomonas reinhardtii (CrHydA1) have been able to shift the steady-state equilibrium of the H-cluster to various intermediate states through different reduction treatments. The

reduction with H2 versus sodium dithionite (NaDT) is shwon to produce both common and unique intermediate states. The hypothesized pathways with intermediate H-cluster states shown in Figure 1.5 illus- trate how complex coordination of the [4Fe4S] subcluster with the [FeFe] reaction site is used during catalysis [12]. Understanding the necessity of each intermediate states is another step towards the creation of useable devices.

1.2 Hydrogenases: uses and challenges in devices

The creation of various biomimetic, bio-inspired, or biohybrid devices that utilize hydro- genases or the natural mechanisms involved is the holy grail of hydrogen-based renewable energy solutions and has been extensively investigated [13, 14]. A major challenge to over- come is the irreversible deactivation of hydrogenases due to oxygen exposure[15]. [NiFe]- hydrogenases are typically more oxygen tolerant but have a catalytic bias and turnover rates that favor hydrogen oxidation. [FeFe]-hydrogenases are much more oxygen intolerant but have the highest turnover rates theoretically approaching 21, 000s−1 with only a 130 mV overpotential [16]. This makes the study of [FeFe] hydrogenases preferable but more prob-

6 Figure 1.4: Calculated catalytic intermediate steps for [FeFe]-hydrogenases. Copied with permission from Greco et al. [11]. Copyright 2007 American Chemical Society.

7 Figure 1.5: Hypothetical intermediate state of the H-cluster upon reduction with NaDT or H2 sparging. Copied with permission from Mulder et al. [12]. Copyright 2013 American Chemical Society.

8 lematic as any amount of oxygen contamination will irreversibly cause proteins to become inactive. Another major challenge in using hydrogenases in biohybrid devices is the pertur- bation of the active site in the process of immobilization onto electrodes. The creation of an efficient biohybrid fuel cell depends on the ability maintain direct electron transfer between

hydrogenases and an electrode which is best achieved if the protein is electrostatically or co- valently immobilized as in Figure 1.6. Evidence suggests that either the process involved to

Figure 1.6: Cartoon of hydrogenase covalently immobilized on an electrode via an organic intermediate. Copied with permission from Baffert et al. [8]. Copyright 2012 American Chemical Society

immobilize a hydrogenase or the state of being immobilized itself negatively affects turnover frequencies [8, 17, 18]. Other sources claim that “an electrode that gently interacts with and quickly transfers electrons to an enzyme should mimic its natural redox partner” allowing

hydrogenases to be immobilized without disrupting their catalytic activity[8]. How the ac- tive site structure differs when immobilized on “gently” interacting electrodes versus others is information that is essential for understanding why certain immobilization strategies are better than others. Any future biohybrid devices will rely on the transfer of electrons to

hydrogenases through a surface that must have optimal biocompatibility and thus the abil- ity to engineer interfaces that do not inhibit but rather facilitate enzyme function will be of utmost importance.

1.3 Objectives

The objectives of this work were to develop novel IR spectroelectrochemical methods to characterize vibrational modes of [FeFe] hydrogenases both in solution and as adsorbed films

9 on crystalline and nanostructured substrates while using available protein in the most effi- cient manner. The limitations on available protein is a significant factor in the determination of which novel methods are most practical. An evolution of techniques was explored wherein the results obtained from a more simple technique was used to improve more complex tech- niques. The first technique is the optical coupling of Si wafers to a ZnSe attenuated total reflectance (ATR) prism which is itself a tool for exploring other methods and materials more quickly and without risk of damage to expensive ATR prisms. Next, functionalized doped Si electrodes was explored as the most simple and robust electrochemical configuration. Func- tionalized evaporated Au films was also studied and combined with doped Si electrodes to make biocompatible Au coated doped Si electrodes. With these, the basic requirements for a spectroelectrochemical cell are expected to be fulfilled after which electrolessly deposited Au and nanoporous Au are to be explored for potential surface enhanced infrared absorbance (SEIRA) properties. The ability to enhance infrared absorbance directly on the surface of an electrode is highly conducive to the study of protein structural changes due to immo- bilization. This has been achieved already with chemically deposited Au on single bounce ATR configurations so the goal in this work is to combine the absorbance enhancement due to multiple bounces with the absorbance enhancement due to a nanostructured surface to make a system with unprecedented absorbance enhancement. The use of nanoporous gold leaf (NPGL) films will also be explored as a conductive membrane for the confinement of proteins to the ATR surface removing the need for highly concentrated proteins in a bulk solution. The study the effects of concentrating proteins directly on the ATR surface by drop casting is also a major objective since this provides the highest concentration of proteins at the ATR surface but it is possible the process of drying CaI can disrupt the active site.

1.4 Previous work

The spectroelectrochemistry of hydrogenases has already been studied with certain [FeFe] and [NiFe] hydrogenases. The use of SEIRAS has been used to observe electrochemically induced changes in the active site of [NiFe] hydrogenases from Desulfovibrio vulgaris immo-

10 bilized on functionalized nanostructured gold surfaces using a single reflection ATR setup [18]. The covalent and electrostatic immobilization of hydrogenases was shown to have ad- verse effects on the active site: no change in amide band intensities in the IR spectra but a decrease in active site modes and losses in catalytic activity were observed. Conversely,

the spectroelectrochemical characterization of the H-cluster in CrHydA1 [FeFe] hydrogenases has already been achieved using a Au mesh electrode in a transmission cell [19]. Though this is excellent for obtaining information about the active site in solution, a careful examination of the effects of immobilization is much more difficult. This work attempts to fill in the knowledge gaps left between the above experiment by developing a method to study the

active site structure of CaI confined to a surface. Michael Ratzloff, a previous graduate student at CSM, started this work with spectro- scopic characterization CaI in different states. The oxidized, reduced and CO-inhibited states of CaI were classified in transmission cells to serve as a fingerprint of CaI. The ox-

idized state shown in Figure 1.7 is characterized by the a peak (g) at 2082 cm1 due to the proximal cyanide, a peak (f) at 2070 cm−1 due to the distal cyanide, a peak (e) at 1969 cm−1 due to the proximal carbonyl, a peak (b) at 1945 cm−1 due to the distal carbonyl and a peak (a) at 1800cm−1 due to the bridging carbonyl.

Figure 1.7: Oxidized spectrum of CaI with vibrational modes corresponding to the following: (a) bridging carbonyl; (b) distal carbonyl; (e) proximal carbonyl, (f) distal cyanide, (g) proximal cyanide. Copied with permission from Ratzloff [20].

11 The CO-inhibited state shown in Figure 1.8 still contain the proximal and distal cyanide peaks (g,f) but shifted to 2075 cm−1 and 2090 cm−1 respectively. The most significant change in the spectrum is due to exogenous CO which is attached to the vacant binding site of the distal iron and interacts with the already present CO ligand to create symmetric and

antisymmetric vibrational modes. The peak (d) at 2016 cm−1 is due to symmetric modes and the peak (c) at 1973cm−1 is due to antisymmetric modes. The proximal carbonyl ligand and bridging CO are mostly unaffected with their peak shift only slightly to 1967cm−1 and 1806 cm−1 respectively.

Figure 1.8: CO-inhibited spectrum of CaI with vibrational modes corresponding to the fol- lowing: (a) bridging carbonyl; (c) symmetric proximal carbonyls; (d) anti-symmetric proxi- mal carbonyls; (e) proximal carbonyl; (f) distal cyanide, (g) proximal cyanide. Copied with permission from Ratzloff [20].

The reduced state shown in Figure 1.9 is from CaI treated with sodium dithionite and hydrogen gas sparging. The spectrum is thought to also contain traces of oxidized CaI as well given that it’s strongest peak at 1945 cm−1 is still present. The bridging CO ligand is thought to separate from the proximal iron forming couples with the other distal carbonyl giving rise to peaks (c) at 1855 cm−1 and (d) at 1898cm−1. The unassigned peak (h) at 1983 cm−1 is uncommon in Clostridial hydrogenases and was previously only reported with Desulfovirio hydrogenases [21] but were thought to be due to intermediate modes in the formation of hydrogen.

12 Figure 1.9: Reduced spectrum of CaI with vibrational modes corresponding to the follow- ing: (c,d,[b]) proximal carbonyls; (e) proximal carbonyl; (f,[f]) distal cyanide, (g) proximal cyanide. Copied with permission from Ratzloff [20].

Preliminary investigations into the characterization of hydrogenase using ATR was also achieved. A CO-inhibited ferredoxin-HydA1 fusion was injected into a custom made flow cell clamped onto the ATR prism at ≈ 8 − 10mg/mL resulting in the spectrum shown in Figure 1.10. Preliminary studies on the effects of protein absorption due to surface

Figure 1.10: CO-inhibited spectrum of CaI using ATR. Copied with permission from Ratzloff [20]. modification of a Si ATR were done using myoglobin. Myoglobin is similar to hydrogenases in that it has an iron-based heme group at its center which when CO-inhibited has a single peak at 1945cm−1. Myoglobin is also not as oxygen intolerant as hydrogenases making

13 carboxymyoglobin (CO-inhibited myoglobin) an excellent test protein. Carboxymyoglobin is also extensively used in this thesis as a cheap and easy way of testing novel methods and materials before using hydrogenase. Where Michael had left off and I began was after finding that traces of the heme site in carboxymyoglobin could not be detected after allowing proteins in solution to adsorb onto a butyraldehyde terminated surface indicating that adsorption had denatured the proteins or that there was insufficient protein adsorbed to be detected.

14 CHAPTER 2 METHODS AND MATERIALS

Infrared spectroscopy, in general, uses the coupling of infrared light to vibrational modes in molecular species to provide a spectrum which correlates to the structure of the species.

Fourier transform infrared (FTIR) spectrometers utilize a polychromatic IR beam from a Globar source in a Michelson interferometer configuration. From a polychromatic source the IR beam is split with one beam reflected back by a fixed mirror and the other reflected back by a moving mirror. The two beams interfere with each other, and after being sent through a sample area are detected with a liquid-nitrogen cooled mercury-cadmium-telluride (MCT) detector. Changes in mirror position create differences in measured beam intensity as a result of different path lengths. The resulting interferogram is Fourier transformed (typically into spectroscopic wavenumbers with units of cm−1) to create a spectrum. Spectral datum is usually collected over several cycles and averaged to increase signal-to-noise ratios at the cost of total acquisition time [22]. FTIR spectra presented in this work were recorded using one of two identical Nicolet 6700 FTIR Spectrometers (Thermo Fisher Scientific) at either the Colorado School of Mines (CSM) or the National Renewable Energy Laboratory (NREL). Data was collected using OMNIC software. Absorbance spectra were obtained by subtracting a known background spectrum from sample single beam spectra on a log scale. All spectra had water vapor manually subtracted using previously collected spectra of water vapor. In addition, all absorbance baselines were fit using a manually adjusted spline.

2.1 Transmission cells

Transmission cells were used for basic FTIR spectroscopy as a standard to compare against other spectroscopic methods. All transmission data involving proteins were taken at NREL using an airtight cell. Transmission cells used two CaF2windows separated by a nylon spacer (Figure 2.1). Proteins, usually in a buffered solution, were dropped into the

15 area between the two windows. The sample volume of protein solution dropped onto the transmission cell was about 10 µL. To avoid oxygen contamination, all samples were prepared

in an MBraun Labmaster SP anaerobic glove box purged with 100% N2 boiled off from liquid nitrogen. Once sealed, the cell is quickly placed inside the path of the IR beam. The sample

chamber is purged with N2 gas which has been passed through a miniature desiccant air dryer (Twin Tower Engineering) and desiccant tube filled with Drierite to minimize water vapor signal and oxygen contamination. All spectra collected at NREL consisted of 512 scans at 2 cm−1 resolution.

Figure 2.1: Passage of IR beam through a contained sample volume

2.2 Attenuated total reflection setup

Attenuated total reflection (ATR) spectroscopy was employed in a variety of ways. Cer- tain novel methods explored in this thesis depended on the properties of ATR prisms and other materials thus an understanding of the optics involved is necessary. From Snell’s Law,

16 there exists a critical angle for photons transitioning from an initial medium to a medium of lower refractive index where a beam is totally internally reflected,

n2 θc = arcsin n1

where n1 is the refractive index of the ATR prism, n2 is the refractive index of the material external to the ATR prism. An optical component that exhibits total internal reflection is known as an internal reflection element (IRE) but will be most commonly referred to in this work as an ATR prism. While the beam is kept within an ATR prism, electromagnetic boundary conditions at the reflecting plane still must be satisfied and thus an electrical

field is created that extends perpendicularly beyond the reflection plane. The field decays exponentially away from the reflection plane and is thus known as an evanescent wave which goes as,

−z/dp E = E0e

where E0 is field strength as the surface, z is the distance from the surface and dp is the penetration depth. The penetration depth is mathematically defined as

λ dp = 2 2 2πn1psin θ − (n2/n1)

where θ is the angle of incidence and λ is the wavelength of the incident beam [23]. The ATR accessory used at CSM is a Research Grade Vertical Variable-Angle ATR (CIC photonics, Inc). Silicon and zinc selenide crystals served as internal reflection elements both measuring 7.9mm by 76.2 mm on each reflecting surface. The IR beam enters one side of the prism and is totally internally reflected bouncing multiple times from each side of the prism.

Due to the design of ATR stage the actual angle of incidence at the reflections plane is limited by the angle at which the IR beam passes into the ATR prism as shown in Figure 2.2(a).

17 Typically the best data was acquired with the ATR accessory set to 35° corresponding to an

angle of incidence on the reflection plane of 40.9° and 10 bounces. Otherwise the stage and

angle of incidence were 45° which allowed 8 to 9 bounces. The penetration depths of this angle range is shown in Figure 2.2(b).

Figure 2.2: Limitations of angle of incidence (θi) and how angle affects both the number of reflections and the penetration depth.

The opposite reflecting plane is pressed against aluminum foil and is assumed to not contribute to any spectra. Depending on the needs of the experiment, spectra collected using ATR consisted of a variety of scans, resolutions and angles. Some experiments were conducted using a horizontal ATR setup at NREL which is a 10 bounce 4 mm by 80 mm

prism with a 45° angle of incidence at the reflection plane.

2.3 Flow cells

The use of a device which would allow the containment and exchange of fluids on the surface of the ATR prism was necessary to perform electrochemistry and spectroscopy si-

multaneously. Two types of spectroelectrochemical (SEC) flow cells with varying sizes were used depending on the needs of the experiment at hand; a small volume cell and a large volume cell containing≈ 330 µL and ≈ 2.5 mL, respectively. Due to the size constraints of the flow cell (shown in figure Figure 2.3) a typical wet reference electrode was not feasible.

Instead, both cells have two 0.045 mm thick platinum wires that span the length of the flow

18 cell running parallel to the IRE reflection plane and each other. These serve as the reference and counter electrodes in a three electrode configuration which is described in more detail in 2.5.

Figure 2.3: Dimensions of flow cell

2.4 Optically Coupled wafers

Since total internal reflection only occurs beyond the critical angle it is possible to use a high index coupling fluid to allow an IR beam to pass beyond the usual reflection plane of the ATR prism into another high index material such as a silicon wafer which can serve as an extended reflection plan. The purpose of engineering such a system is to allow the rapid testing of wafers as though they were ATR prism surfaces. Ideally, using this optical coupling method, functionalizations requiring long depositions could be carried out on many different Si wafers simultaneously and then tested in quick succession potentially saving

19 several days to week worth of waiting. The setup shown in Figure 2.4 within this work is called an ATR/wafer stack. Coupling fluid was applied by spreading the solution along the length of the ATR prism with care taken to make sure no bubbles formed. A sample wafer cut to approximately match the dimensions of the ATR surface was then placed on

top. Excess coupling fluid was absorbed with a kimwipe or q-tip and then a flow cell was clamped against the ATR/wafer stack. After experiments were completed, the ATR was sonicated with methanol.

Figure 2.4: Silicon wafer with ideal optical coupling to ZnSe ATR

Most high index coupling fluids have many IR modes that tend to obscure peaks of interest from a species in the flow cell. Diiodomethane, with a refractive index of 1.77, was chosen as the best coupling fluid since it’s relatively simple molecular structure gives rise to the least IR modes. Diiodomethane, however, was found to crystallize around the wafers edges and over the course of few hours would no longer serve as a coupling fluid. Thus experiments with coupling fluid were conducted as quickly as possible to avoid the degradation of the coupling fluid from affecting results. As an alternative, Cargille MeltmountTM, which is a thermoplastic with a high refractive index, was used in certain experiments. The procedure

for applying Meltmount is somewhat extensive since the material must be heated above 80 °C

to transition to a liquid phase. The Meltmount was usually heated in bulk to slightly above

80 °C in a water bath. Once fluid, the Meltmount was poured onto the backside of a wafer sample and allowed to solidify. The wafer with Meltmount was then placed on a ZnSe ATR

20 and the flow cell was used to loosely clamp the assembly together. With the solid Meltmount under slight pressure between the ZnSe prism and Si wafer, the entire assembly was reheated

to about 80 °C. Once the Meltmount was in liquid phase again, the assembly was tightened until the wafer appeared completely compressed against the ATR prism. The assembly was

allowed to cool before placement inside the spectrometer.

2.5 Electrochemical setup

A standard three electrode configuration was used for all electrochemical experiments. Of the two platinum wires implanted in each flow cell, one was used as a reference electrode while the other was used as a counter electrode. The use of platinum as the reference creates a quasi-reference electrode system in which the potential relative to a standard hydrogen electrode (SHE) will vary with pH as well as have a large constant shift. Cyclic voltam- mograms of the redox of ferri/ferrocyanide was used as an internal standard for each new material or method explored since the observed potential would shift by several hundred millivolts vs. SHE. Observed potentials would remain fairly consistent with respect to each type of material or method rarely straying more than 20 mV. A Model 173 Potentiostat with a Model 176 Current-to-Voltage converter (EG&G Princeton Applied Research) was used to control electrochemical potential within the flow cell. An e-corder 210 data acquisition unit (eDAQ model ED210) and corresponding software was used to record cyclic voltammetry data. Details about working electrodes will be discussed in detail in 2.9. Solutions of 100 mM phosphate buffers of varying pH were made from standard buffer capsules(Cole-Parmer In- struments Co) added to 100 mL DI water to make phosphate buffered salines (PBS). Oth- erwise a solution of 50 mM Tris buffer (pH 8.0) with 100 mM NaCl and 5% glycerol was used.

2.6 Rapid electrochemical modulation

Rapid electrochemical modulation is a method in which an analyte is oxidized and reduced repeatedly in quick succession with spectra taken between each step allowing for any artifacts

21 in the spectrum that may change over time, such as water vapor or coupling fluid, to easily be subtracted out. The standard procedure for obtaining redox difference spectra was as follows were IR spectra are taken after each step:

1. Inject buffer solution

2. Inject analyte solution

3. Set the potentiostat below reduction potential of analyte

4. Set the potentiostat above the oxidation potential

5. Repeat steps 4 and 5 several times.

6. Subtract average of oxidized spectra from average of reduced spectra

If we define a series of spectra as such:

S1 = A(−)+ B(t1)+ C(t1)

S2 = A(+) + B(t2)+ C(t2)

S3 = A(−)+ B(t3)+ C(t3)

Where A(−) is the contribution to the spectrum due to a reduced species, B(t) is the contri- bution due to adsorbed species, and C(t) is the contribution due to changes in the coupling medium. I assume that the voltage, time, and wavenumber portions of each spectrum are independent of one another. The adsorption kinetics of an analyte such as proteins can be modeled as a reaction involving surface sites that fill up over time. A simple expression such

−t/τ as B(t)= B0(1 − e ) (where t is time and τ is a time constant) would be appropriate but

22 for any analyte we need only consider two situations. Either the analyte is freshly injected and is just starting to accumulate on the surface or the analyte is mostly in equilibrium.

By applying a first order expansion of the function B(t) B0t/τ for t ≪ τ and B(t) B0 for t ≫ τ. The change in coupling fluid spectra tends to be linear thus C(t) = C0t. Assuming t(n+1) − tn = tn − t(n − 1) (periods between scans are equal) then ∆B and ∆C are close enough to the same between each spectrum to be equal for all intervals. Thus:

2S2 − S1 − S3 = 2∆A

S3 − S1 = ∆B + ∆C

This shows that by using rapid electrochemical modulation and the above formula, one can rather accurately separate the redox difference spectra from the changing background. In most experiments the time to take spectra with 200 scans (166 s) allowed for sufficiently rapid

modulation. Results improve by averaging over several oxidation/reduction spectra. While this works fine when the coupling medium changes linearly with time, some media change in different ways over similar time periods thus it is difficult to determine what changes in spectra are due to the state of the analyte and not the coupling medium. Ideally, species

directly on the surface of the working electrode should change immediately and thus any second order variation of the coupling medium can be neglected

2.7 Functionalizations of Silicon

Silicon surfaces whether they were wafers or prisms were functionalized with various self- assembled monolayers (SAMs). Prior to all silanization procedures the substrate was cleaned

in 3:1 mixture of concentrated sulfuric acid and hydrogen peroxide known as piranha for 10 − 15 min. Typically the samples were also immersed in 10% hydrofluoric acid (HF) to strip the native oxide then re-immersed in piranha for 2 min to regrow a thin oxide layer

23 of consistent thickness. Monolayers of triethoxysilylbutyraldehyde (TESBA) were obtained by immersing cleaned samples in a 1% v/v solution in ethanol for 40 min. Monolayers of 3-aminopropyltriethoxysilane (APTES) were obtained by immersing samples in a 0.5% v/v solution in Toluene for 1 hr. Monolayers of carboxyethylsilanetriol salt (CSS) were obtained by immersing samples in a 0.5% v/v aqueous solution with pH adjusted to 4 with hydrochloric acid (HCl) for 40 min. Monolayers were characterized by water contact angle measurements and ellipsometry. Further details about the fabrication and characterization of CSS monolayer in particular are provided in appendix A.5.

2.8 Preparation of Proteins

Due to the extreme sensitivity of CaI to oxygen, myoglobin (Mb) from equine heart (SDS-PAGE, Sigma) was often used as a test protein. The spectroelectrochemical oxidation and reduction of myoglobin has been shown [24, 25] and the absorbance spectrum of CO- inhibited myoglobin [26] provide an easy way to test novel spectroscopic methods such as optically coupled wafers. Myoglobin is a smaller protein with mass of only 17 kD compared to the typical mass of CaI at ≈ 65 kD. The active site equivalent for myoglobin is a single heme group, shown in Figure 2.5, and it is the ligation of the central iron with a carbonyl that results in a distinct IR mode at 1945 cm−1.

Mb was stored at −10 °C as a lyophilised powder. To obtain CO inhibited Mb in solution,

sodium dithionite (NaDT) was used to reduce Mb and scavenge the oxygen ligand from the

heme site. NaDT powder was put in a vial and purged with N2 for 5 − 10 min then PBS was injected to the vial using an air-tight syringe such that the final concentration was

≈ 280 mM. Mb powder would be purged with N2 in a separate vial and NaDT solution would be transferred via air-tight syringe. Vials were gently mixed so as to not disrupt the protein. CO was bubbled through the Mb solution taking care to minimized air exposure. During the phase of experimentation where Mb was scanned in solution phase, pure CO from a lecture bottle was used.

24 Figure 2.5: Structure of Oxymyoglobin and a close-up view of the heme site. Image of 1MBO Philips [27] created with Protein Workshop from Moreland et al. [28].

In later experiments, dried protein films were found to be a more efficient use of protein since this would allow all proteins to be confined within the penetration depth of evanescent waves. Protein films were created by drop cast of a dilute protein solution onto a sample surface such that a single long drop that matched shape of the flow cell O-ring. In some experiments the particular volume and concentration of drop cast protein solutions varied and thus only a total protein mass and dry time rather than concentration will be reported in this work. Often, Mb films were put in a transparent box with a low flow of N2 to make the films dry faster. For experiment involving the CO inhibition of Mb films, a solution of

anaerobic NaDT would be made and then saturated with CO. During these experiments, CO was chemically generated by the dehydration of formic acid in heated sulfuric acid. The resulting CO gas was bubbled through DI water before entering any vial containing solution used in experiments. Formic acid was added at a constant rate using a Model 100 KD

Scientific syringe pump set to 50 mL/hr. All CaI solutions were initially received from NREL in 50 mM Tris buffer (pH 8.0) with 100 mM NaCl (ACS reagent, Sigma) and 5% glycerol (molecular biology, Sigma-Aldrich. Occasionally CaI in solution without glycerol was desired in which case the protein was

passed over a G-25 column and equilibrated with a solution of 50 mM Tris buffer (pH 8.0)

25 with 100 mM NaCl. For all experiments with CaI, the spectrum of the starting solution in

its as-prepared (Has-prep) state was collected to verify the current batch had detectable active site modes. While drying, all hydrogenase films were stored in an anaerobic refrigerator at

4 °C.

2.9 Working electrode fabrication

The characteristics of the working electrode were of particular interest during this work. The working electrode regardless of its material or structure needed to be able to support a range of applied potentials repeatedly without tarnishing, must have low sheet resistance, and be able to be functionalized with relative ease. Working electrodes that sufficiently met these characteristics are discussed in detail in the results chapter. Other materials used as working electrodes but could not be optimized to be used in further experiments are discussed in detail in the appendices.

2.9.1 Semi-conductor electrodes: Doped silicon

With a sufficiently doped layer a Si wafer can provide both enough conductivity to suf-

fice as a working electrode while still remaining transparent in the infrared. A doped layer with a high carrier concentration will have an increased plasma frequency making it behave like a metal and reflecting infrared light coming from the bulk Si back into the bulk before reaching the surface. The concentration of carriers decreases as a function of depth thus carrier concentration was tuned by etching away portions of the doped layer. The process of fabricating doped Si layers with low sheet resistances but still transparent in the IR is explained in detail in A.4. A problem with using doped Si as an electrode is due to the nature of semiconductor-electrolyte interfaces. The density of states within silicon limit the range of potentials at which the charge transfer will occur quickly. A highly doped n-type semiconductor will have a Fermi energy closer to its conduction band. When the semicon- ductor comes in contact with an electrolyte it experiences band bending and the region near the interface will donate free carriers until the Fermi energy of the semiconductor equals

26 the Fermi level of the redox couple in the electrolyte as shown in Figure 2.6. The region where the bands bend (the space charge or depletion region) depend on the carrier concen- tration.This simple depiction does not take into account surface states, photoexcitations, or effects of recombination of electrons with oxidized species but it does show that, in general,

the interface will have rectifying characteristics. Changing the potential of the doped wafer will still change the chemical potential of the solution such that the analyte will be reduced or oxidized once a state for an electron or hole respectively is available [29].

(a) Energy levels of a semiconductor and an electrolyte with a(b) Semiconductor electrolyte interface at equilib- redox couple rium

Figure 2.6: N-type Semiconductor-Electrolyte Interface at Equilibrium.

To make a doped layer P8545 spin-on dopant was spun onto double-side polished wafers using a G3P-8 Spin Coater at 3000 rpm for thirty seconds. Wafers were put in a diffusion oven set to heat to 1250 ◦C for thirty minutes. Later analysis suggests the oven did not achieve this temperature but wafers were sufficiently doped for further tests. Wafers were immersed in 10% HF and characterized via 4-point probe. Sheet resistances were about 3.1 Ω/sq which

corresponds with a diffusion depth of only about 4.3 ñm. The high carrier concentration in the doped layer is enough to make the silicon behave metallic for infrared wavelengths. An

SF6 plasma was used to etch layers of the wafer away. Complete transmission for wavenum- bers above about 650cm−1 was achieved with no degradation of electrochemical capability.

27 Out of two Si ATR prisms available only one was heavily phosphorus doped while the other was left for control experiments. The doped Si ATR was etched until spectroscopic baseline returned to nominal levels. Although the final sheet resistance is relatively high (∼ 156.5 Ω/sq) this was sufficient to oxidize or reduce an analyte.

2.9.2 Evaporated gold thin-films

Evaporated Au films were used since their use as biocompatible electrodes is well understood[30– 32]. Planar Au film electrodes are useful as the surfaces do not easily oxidize and can be used as a stable working electrode given that it is adhered well to a substrate. During the course of this thesis, evaporated Au thick-films (≥ 50 nm) were originally used as control groups to compare against experimental electrodes such as Au thin-films (3 − 5 nm) and are discussed in detail in appendix A.3. Thin-films discussed in the main body were fabricated by cleaning double side polished Si wafers in piranha solution then hydrogen terminating the surface by immersing in 10% HF. Hydrogen terminated wafers were quickly transferred to an Edwards

Diffusion Pumped Evaporator and pumped down to ≈ 2 ∗ 10−6 Torr. To improve adhesion and uniformity of the Au film, 4 − 6 nm of Cr is evaporated followed by 3 − 5nm of Au from Tungsten boats (R.D. Mathes) in succession without breaking vacuum [30]. Au films were often functionalized with 3-mercaptopropionic acid (MPA) which have a short alkyl chain terminated with a thiol on one end and a carboxyl group on the other

end. MPA functionalized surfaces have been shown to electrostatically immobilize proteins such as hydrogenase [17, 33, 34]. The method for forming good monolayers of MPA requires at least twelve hours of immersion in ≈ 1 mM MPA (in ethanol) [35, 36]. Depositions on sufficiently thick Au films were verified with polarization modulated infrared reflectance

absorbance spectroscopy (PM-IRRAS) by taking scans of the wafers before deposition and then scanning again after ∼ 19 hrs of immersion in MPA.

28 2.9.3 Nanoporous gold leaf

Nanoporous gold leaf (NPGL) is a material that has yet to used in spectroelectrochem- istry but works incredibly well as a electrode and potentially has infrared absorbance en- hancing effects. NPGL films were created by floating 100 nm thick Monarch 12 Karat white gold (fineartstore.com), which is a Au-Ag alloy, in concentrated nitric acid for various times [37] which dissolves most of the silver within the alloy leaving only a porous Au network. The NPGL is transferred and floated on DI water to quench dealloying. The possibility of infrared absorbance enhancement near the surface of the NPGL was partially investigated but cut short due to time constraints. The details of these investigations are covered in appendix A.7. NPGL films were still used in various configurations as a conductive filter. Transferring the NPGL films on top of dried protein films was possible by quickly immersing the protein film and substrate under water and quickly covering with the NPGL films. If done carefully, a majority of the protein film would remain and the NPGL would form a continuous cover. Alternatively, drying NPGL films on weighing paper allow the films to be positioned over dried protein films without losing any loosely bound proteins on the surface. Films were usually etched for 35 min resulting in an average pore diameter of about 40nm as determined by SEM images like in Figure 2.7. The dimensions of myoglobin (Mb) are that of a 4.3 nm by 3.5 nm by 2.3 nm disk [38]. While Mb is small enough to fit into the pore of 35 min etched NPGL its diffusion rate is much smaller than that of molecular species such as dithionite ions or carbon monoxide gas. For the standard duration of any experiment performed with NPGL films any protein is considered trapped but can still interact with chemical species injected into a flow cell. For experiments using NPGL dried on paper, protein films were dried without glycerol since films with glycerol would tend to spread out past the ATR prism when the NPGL/paper was put on top.

29 Figure 2.7: SEM image of NPGL etched for 35 minutes in concentrated nitric acid courtesy of Peter Ciesielski.

30 CHAPTER 3 RESULTS AND DISCUSSION

3.1 Scope and Constraints of This Study

It is important to make note of the particular constraints encountered during this work which were a major factor in exploring novel methods. The very nature of using FTIR spectroscopy to study aqueous solutions is limited by the fact that water can completely absorb all IR light within a problematically large spectral range. For this reason ATR-FTIR spectroscopy is necessary so that species in solution have a chance at being detected in bulk solutions. Furthermore, the extensive process necessary to acquire any significant amount of CaI makes it incredibly precious for experiments, and it must be used as conservatively as possible. Even once hydrogenase is available the vibrational modes of interest are merely the few ligands buried within the protein and increasing the number of proteins within an evanescent wave is necessary to obtain significant details about the active site. The maximum concentration of proteins is limited by the fact that the solution becomes so viscous it cannot be injected to the flow cell [20] so methods to either enhance IR absorbance of the active site or increase protein density at the ATR surface were developed out of necessity. In addition the flow cell and ATR prisms had to be modified to allow electrons to be pumped into hydrogenases near or on the ATR surface. Therefore, an additional constraint was that any spectroscopic method had to be compatible with electrochemical needs. For this reason doped Si, Au films, and even Au coated doped Si were characterized and tuned to balance IR transmission and conductivity. Some techniques would satisfy all experimental constraints but the processes needed to fabricate certain elements were deemed too extreme to apply to the delicate and expensive ATR prisms thus a way of optically coupling cheap and abundant Si wafers was devised and found useful as a technique in itself. Present throughout all of these techniques is the need to maintain anaerobic conditions and verifying that any

31 new method does not disrupt protein function. The world of IR spectroelectrochemical method development given these constraints is like exploring a series of 1 m wide caves; the possibilities are endless yet there is still very little room to maneuver. In the sections below, the results of a kind of evolution are given where techniques were tweaked or combined until

it was seen to have some advantage over the others. As with organisms, techniques that evolved later were not necessarily superior overall but simply provided an advantage over other techniques in a particular way.

3.2 Optically Coupled Wafers

In the course of conducting experiments involving Si ATR prisms some surface func- tionalizations or treatments may be avoided simply out of concern for the integrity of the ATR prism which is usually delicate and costly to replace. It is preferable to conduct tests

using Si wafers but this comes at the cost of using ATR spectroscopy thus a way to test Si treatments on wafers and optically coupling them to an ATR was collaboratively developed. Taylor Angle, a colleague at CSM, calculated that there should be a range of incident angle in which the IR beam would pass beyond ATR into a Si wafer and reflect off the Si wafer back into the ATR. To demonstrate as proof of concept that Si wafers could be optically

coupled to a ZnSe ATR prism allowing total reflection to occur at the Si surface, spectra of

4% v/v MPA in ethanol were taken using an ethanol background with the ATR stage set at

° ° 45° and ≈ 52 . With the stage at 45 the IR beam should pass into the wafer and be reflected

at its surface showing a clear MPA spectra. With the stage set to 52 ° the IR beam would

reflect off the ZnSe/coupling fluid interface resulting in a spectrum with no MPA peaks. To maintain a constant thickness of the coupling fluid between the wafer and the ATR prism, 5 µm spacers were mixed with the coupling fluid. Figure 3.1 shows that MPA is detectable within the appropriate angle range and not detectable outside of the appropriate angle range

proving the concept. The use of coupling fluid without spacers, however, produces interesting results. Since the flow cell and thus wafer are tightly clamped onto the ATR prism, the space between the

32 Figure 3.1: (Left) Spectra of 4% MPA in ethanol taken using wafers with the angle of incidence set within calculated angle for optical coupling (purple) compared to angle of incidence set outside of calculated angle for optical coupling (yellow). Spectrum of 1% aqueous MPA (5x scale) provide for reference (blue). (Right) Illustration of setup with spacers. wafer and the coupling fluid can become small enough for frustrated total internal reflection to occur wherein a significant fraction of the IR beam can transmit through the coupling fluid layer even when the angle of incidence is beyond the critical angle for total internal reflection at the ZnSe/coupling fluid interface. Repeating the above experiment without using spacers produces the data shown in Figure 3.2. In addition to an increase in the MPA

peaks detected with the ATR stage set to 45° a nearly equal MPA spectrum is obtained

with the ATR stage set to 52°. Optical coupling fluid with spacers allows for total internal reflection at two distinct interfaces using the stage angle as a switch for probing the flow cell. Precisely switching between angles would allow for a form of dual reflection plane modulation spectroscopy which could be used as a way to detect species on the surface of a

Si wafer without a reference spectrum before said species was added to the surface. In this work a reference spectrum was usually taken so the use of coupling fluid without spacers was preferred since this appears to improve the strength of absorbance spectra. The use of optically coupled Si wafers was not without drawbacks. Over the course of longer experiments the coupling fluid will appear to dry out and give rise to new or shifted

33 Figure 3.2: (Left) Spectra of 4% MPA in ethanol taken using wafers with the angle of incidence set within calculated angle for optical coupling (purple) compared to angle of incidence set outside of calculated angle for optical coupling (yellow). Spectrum of 1% aqueous MPA (5x scale)provide for reference (blue). (Right) Illustration of setup without spacers. peaks over most regions of interest. By taking spectra of just a blank wafer optically coupled to a ZnSe ATR prism, various peaks rise, fall and shift seemingly independent of one another. The difference spectrum as shown in Figure 3.3 demonstrates how the coupling fluid changes between 10 min increments over an hour. The magnitude of these changes is two to three orders of magnitude greater than any spectral peaks Although the changes in coupling fluid modes present problems the technique itself is clearly useful for ATR-FTIR spectroscopy. Use of optically coupled wafers was used as a tool for studying various electrode materials and configurations described in the sections below.

3.3 Doped Si electrode

Doped Si provides the most robust electrode capable of being reused and going to extreme potentials that tended to destroy other electrode surfaces. The process of engineering doped

Si layers to be transmissive in the IR yet still conductive was tested using wafers and, through the convenience of the optical coupling method, used for initial spectroelectochemical experiments as specified in appendix A.4. The most optically simple configuration for ATR

34 Figure 3.3: Difference spectra of diiodomethane coupling fluid after (a) 15 min, (b) 30 min, (c) 45 min, (d) 60 min, (e) 75 min. Each spectrum uses the previously taken spectrum as its background.

35 based spectroelectrochemistry was to directly dope the surface of a Si ATR prism and this was done once the process had been refined through testing with optically coupled doped wafers. To verify the doped Si ATR served as sufficient working electrode and optical element the spectra of both oxidized and reduce methyl viologen (MV) was obtained. A solution of

10 mM MV in Tris buffer was injected with the large volume flow cell sealed against the doped Si ATR. The CV of MV is shown in Figure 3.4 along with a CV of Tris buffer as a baseline.

Figure 3.4: Cyclic Voltammograms of 10 mM Methyl Viologen in Tris buffer sweeping from −1.4V to 0V at 10mV/s.

Spectra were recorded with the potential held at −1100 mV to reduce MV and then at −100 mV to oxidize MV. As shown in Figure 3.5 the doped Si ATR is initially able to oxidize and reduce MV. With each iteration there is less change in the spectra and eventually the state of MV cannot detectably be changed. This is likely due to a build up of a native oxide layer on the ATR surface. Functionalization of the silicon surface will prevent oxide growth, however, the forma- tion of organic monolayer directly on an oxide free Si surface typically requires extensive chemical synthesis and the use of argon-based free radicals [39, 40]. It has been shown that a sufficiently thin oxide layer can be made to minimize resistance between the doped

36 Figure 3.5: 10 mM Methyl Viologen in Tris buffer oxidized at −1100 mV and reduced at −100 mV in succession with doped Si ATR. layer and electrolyte while still allowing siloxane-based SAMs to form [41]. To see if such an electrode could be made to work with spectroelectrochemical setup in this work, doped silicon wafers were functionalized with APTES and optically coupled to a ZnSe ATR prism via Meltmount. A solution of 10 mM ferricyanide in pH 6 PBS was used as an analyte. Voltammograms cycling between −700mV and 600mV at 100mV/s shown in Figure 3.6(b) appear only to oxidize the ferricyanide, however, the oxidation peak consistently appears after several cycles indicating that reduction must also be taking place even if it isn’t clear from voltammetry. This is further evidenced by the consistent oxidized-minus-reduced spec- tra in Figure 3.6(a). A peak at 2092cm−1 appears to develop over time indicating that some chemical species may be forming on the surface that is not silicon oxide. The oxidation peak in voltammograms taken after spectroscopic scans has also shifted indicating that the adsorbed species is impeding electrochemical activity but not completely preventing it.

3.4 Au-coated doped Si electrodes

After the development of doped wafers that were IR transparent yet still conductive, the ability to have organic monolayers without an insulating oxide layer was desired. Since the

37 Figure 3.6: Spectroelectrochemical characterization of APTES functionalized, doped Si wafers optically couple to ATR prism: (A) Oxidized-minus-reduced spectra; (b) Cyclic voltammograms. For both graphics blue is initial data, purple is intermediate data, and yellow is final data. grafting of an organic monolayer directly to bare Si was not practical for this work, the use of Au coatings were explored as a way to interface thiol-based SAMs with doped Si. It has been shown that ultra thin Au films can be used as electrodes on the reflecting plane of an ATR [30] however there are several challenges to overcome. The Au film must adhere well to the Si substrate; in electrochemical experiments Au films evaporated on mere Si will lift off the surface beyond a certain range of potentials. Au evaporated on hydrogen terminated

Si tends to adhere well but a very thin film of Cr will increase the wetting of the surface for Au allowing for a more uniform film and adhere the Au film well enough to withstand the range of potentials applied for most electrochemical experiments. A Cr thin-film itself forms continuous layers even when only 2 nm [42]. For calibration and initial testing purposes, thick

Au coatings (≈ 200 nm) were evaporated onto non-doped silicon wafers with 5 nm chromium adhesion layers. A solution of 10 mM ferricyanide in pH 4 PBS was used an analyte in an open electrochemical cell. Voltammograms cycling between −800mV and 500mV at 100 mV/s show in Figure 3.7 classic ferri/ferrocyanide redox curves[43].

Since Au thin-films below ≈ 10 nm tended to lack sufficient conductivity to serve as a working electrode, doped Si wafers were used as a substrate to improve conductivity. At such

38 Figure 3.7: Proof of working electrochemical system by demonstration of classic ferri/ferrocyanide cyclic voltammograms. a low thickness it is possible the Au coating is not complete and in this case it is distinctly possible to have a mixed electrode effect, however, this is not as much of a concern when considering that the Au coated doped silicon would ultimately interface with proteins via an organic intermediate. Ideally, any protein close enough to accept an electron directly from the electrode will be immobilized by, for example, an MPA ligand which would be bonded to a Au surface site as illustrated in Figure 3.8.

Figure 3.8: Cartoon of incomplete Au coating with an MPA SAM and immobilized protein.

39 A basic spectroelectrochemical test was conducted to verify this configuration would work. For this experiment, a doped Si wafer was cleaned in piranha solution then immersed in 10% HF for 3 min to hydrogen terminate the surface. Immediately after hydrogen ter- mination, the wafer was transferred to an evaporation chamber in which 4.1 nm of Au was

deposited at ∼ 3 ∗ 10−6 Torr. The wafer was then immersed in a 1% v/v solution of MPA in ethanol for 23 hrs. After rinsing the wafer with ethanol to remove loosely bound thiols the wafer was assembled in an ATR/wafer stack with 1.77 index coupling fluid and the small volume flow cell. After letting water vapor purge from the spectrometer, a solution of 10 mM

K3Fe(CN)6 in pH 6 PBS was injected. The electrode potential was cycled from −400 mV to 400mVat 10mV/s. The resulting voltammograms, shown in Figure 3.9(b), indicate ferri- cyanide reduction and ferrocyanide oxidation occur but with larger overpotentials than Au thick film electrodes. Rapid electrochemical modulation between −400mV and 400mV was employed to obtain a clear reduced-minus-oxidized spectrum shown in Figure 3.9(a). Peak

intensities diminished with each iteration and cyclic voltammograms afterward show no sign of oxidation or reduction, however, the gold film itself appeared to remain intact upon visual inspection after the experiment.

Figure 3.9: (A) Decaying oxidized-minus-reduced spectra of ferri-/ferrocyanide using Au thin-film on a doped silicon wafer with numbers corresponding first, second, or third redox cycle. (B) Initial CV

40 It should be noted that this configuration is electronically very complex and experiments were conducted comparing the behavior of thick Au films with Au coated Si and Au coated doped Si when oxidizing and reducing ferricyanide in buffers with varying pH levels. Since there are multiple interfaces and capacitances to consider not much could be concluded about data gathered other than that the electrodes appear to be able to oxidize and reduce an analyte at some voltage which is, arguably, the only result that matters.

3.5 NPGL films

The decision to investigate NPGL was due to a brief collaboration with Peter Ciesielski at NREL. Since NPGL films are only ≈ 100 nm thick yet still easy to make and adhere to silicon, it seemed worthwhile to test how they would work in a spectroelectrochemical cell. Due to its porous nature NPGL films have been shown to be able to probe the vibrational modes of species both on top of the film and inside the pores [44]. NPGL is highly conductive but appears to still allow IR evanescent waves through. In addition the high surface area would allow for increased adsorption sites for proteins improving catalytic currents and possibly IR absorbance. Thus, the use of NPGL films was expected to have the best characteristics as a working electrode for a spectroelectrochemical cell. To test this, a NPGL film was etched for 35 min and then dried on a ZnSe ATR. Once dry the large volume flow cell was clamped onto the ATR. A solution of 10 mM ferricyanide in pH 6 PBS was electrochemically modulated in the same fashion as described above. Very little variation is seen in oxidize- minus-reduce spectra shown inFigure 3.10(a). The cyclic voltammograms before and after spectral acquisition shown in Figure 3.10(b) have a rather large shift in midpoint potential and a large increase in overpotential. The reason for such large changes in CV’s is unclear. After disassembling the cell most NPGL films appear to have been shredded, however, there has been no experiment involving ferricyanide where the NPGL films could not consistently change the state of the analyte. Although it is possible that the NPGL does not stay intact throughout an entire experiment, it is far more likely that the NPGL films are disrupted by the separation of the flow cell from the ATR after completing an experiment.

41 Figure 3.10: (A) Oxidized-minus-reduced spectra of ferri-/ferrocyanide using NPGL on ZnSe ATR. (B) Corresponding CV’s. For both graphics blue is initial data and yellow is final data.

For reasons better explained in 3.6 a way to cover the ATR surface with NPGL without having to submerge the ATR under water was desired. After trying to change the method little by little the idea to simply dry the NPGL films on weighing paper appeared to work well and actually evolved into it’s own technique. These techniques are particularly useful because it allows for a Au electrode to be adhered or close to the surface of the ZnSe prism which allows for spectral data to be captured below 1500 cm−1. To demonstrate this, a 35 min etched NPGL film was dried on weighing paper. pH 6 PBS was injected into the flow cell and the system was left alone for about 30 min since it appears to take longer for the the IR peaks of water to equilibrate, presumably due to the

fact that is it slowly diffusing into the NPGL pores. Then a solution of 10 mM MV in pH 6 PBS was injected and allowed to equilibrate for about 5 min before collecting voltammograms cycling between −1.1 V and 0 V as shown in Figure 3.11. Once the oxidation and reduction potentials had been verified, the potential was manually modulated between −900 mV and

−700 mV with IR spectra taken after each potential change. The resulting difference spectra are shown in Figure 3.12 with a more complete and distinct set of absorbance peaks for the oxidized and reduced states of MV. Preliminary studies were conducted to see if some form of absorbance enhancement could be observed with NPGL films of various etch times the

42 Figure 3.11: Cyclic voltammogram of methyl viologen using NPGL films as a working elec- trode

details of which are described in A.7.

3.6 Confinement of myoglobin films

Although electrochemical and spectroscopic challenges could be overcome to conduct ba- sic spectroelectrochemical tests, a way to increase the total IR absorbance of proteins within this framework was needed. Significant effort was put into the investigation of electrolessly deposited Au films for SEIRAS as detailed in A.2, however, unexpected complication arose and the technique was deemed impractical for this work. Instead, absorbance was enhanced simply by drying proteins on an ATR surface. With carboxymyoglobin films, a strong and reproducible signal could be detected using much less protein. Table 3.1 shows how the best variations of selected methods compare against the use of protein in solution. Details on evolution of these methods is provided in appendix A.1.

Table 3.1: Comparison of best data with Mb in solution

Method Peak Height/mg (∗103) Mb film on ZnSe ATR with15 min etched NPGL on paper 76.9 Mb film on doped Si ATR with by one day etched NPGL 2.3 Concentrated (80 mg/mL) CO-Mb in solution 0.004

43 Figure 3.12: Spectra of methyl viologen in an (a) oxidized state, (b) reduced, (c) oxidized state again.

44 Although the proteins films are rather stable even when buffers are injected into the flow cell there is still a tendency for the films to diffuse away from the ATR surface and into the bulk electrolyte. Ultimately, a way of confining the protein to the ATR surface while still allowing it to interact with electrolytes in a large volume flow cell was desired. The solution was to use a confining membrane such as NPGL or paper over a protein film allowing a small amount of protein to be scanned at the ATR surface while allowing ample room for the electrolyte solution. Furthermore this method allows a confined protein layer to interact with chemicals injected into the flow cell through the cover. To demonstrate this, 0.25mL of 4mg/mL myoglobin in DI water was drop cast on a ZnSe ATR prism and allowed to dry

overnight. A film of 15 min etched NPGL dried on weighing paper was used to cover the protein film with the large volume flow cell clamped into the ATR assembly. A cartoon of the configuration is shown in Figure 3.13.

Figure 3.13: Cartoon of myoglobin films confined by a NPGL membrane and chemical in- teractions with a bulk solution.

The flow cell was injected with PBS and allowed to equilibrate before injecting a 280 mM solution of NaDT in PBS followed by a CO saturated solution of 280 mM NaDT in PBS.

Once the characteristic Fe-CO peak at 1967 cm−1 reached a maximum peak intensity, PBS equilibrated with ambient oxygen levels was injected at 1 mL/min until the Fe-CO peak almost completely diminished. A CO saturated solution was injected again, allowed to max-

45 imize, then additional oxygenated PBS was injected again. Figure 3.14 shows the repeated CO inhibition of myoglobin films made possible by confinement with NPGL.

Figure 3.14: Confined Mb films repeatedly CO inhibited. CO saturated solutions of NaDT in PBS were injected at time points labeled CO. PBS buffer equilibrated with ambient oxygen levels is injected at 1 mL/min starting at time points labeled O2. Time increments between collected spectra were ≈ 8 min. Any peaks other than the Fe-CO peak at 1967 cm−1 are due to water vapor.

3.7 Affects of drying on CaI films

The use of dehydrated protein films is particularly useful when studying CaI because rel- atively small amounts could be provided by NREL for experimentation. Decreasing the total mass of protein necessary for detection allows for more experiments to be conducted with the amount provided. Thus it became necessary to observe how drying CaI into condensed films affected the active site. Typically CaI from NREL started in a 50 mM Tris buffer (pH 8) with 100 mM NaCl with 5% glycerol. When dried the glycerol would remain creating a

46 thick molasses-like layer that would tend to spread past the ATR prism when a cover was applied. Thus, the effects of drying CaI into films without glycerol was studied to ensure the process was not detrimental to the protein or its active site. The protein in solution phase was scanned both as prepared and then again after transferring the protein to a Tris buffer without glycerol to ensure no significant changes occurred as shown in spectrum (a) and spectrum (b) in Figure 3.15 respectively. A small peak at 1985 cm−1 is seen briefly but is not thought to be significant and the experiment was continued. Two films were created

by drop casting 10 µL of 18mg/mL on CaF2 and letting them dry at 4 °C. One window, as soon as the film appears to be dry enough not to flow off the surface, was assembled in the

transmission cell and scanned every thirty minutes for five hours. The other window was

left at 4 °C in the glove box and scanned after five hours. Changes in the active site after drying CaI films are shown in spectra (c) through (f) in Figure 3.15. CaI films were made on the ZnSe ATR at NREL to verify active site modes could still be detected in an ATR setup. A solution of 250 µL of 2mg/mLCaI was evenly spread across the horizontal ATR prism then allowed to dry for 3.5 hrs. The same test was conducted at Mines on the vertical ATR after transporting the same batch of CaI. Figure 3.16 shows a comparison of the results. In experiments with ATR prisms the sensitivity is still enough to clearly distinguish the strongest peak of the auto-oxidized state at 1950cm−1 but other peaks cannot be distinguished above the noise. The strongest mode seen in spectra collected in the vertical ATR setup is half as intense as peaks in spectra taken with the horizontal ATR setup. The spectra from ATR appear weak when compared to transmission spectra however this represents a 24-fold enhancement, on a per-milligram basis, compared to the CO-inhibited spectrum of hydrogenase taken in solution phase in Figure 1.10. Experiments with CaI films confined by paper were also conducted but in these instances little to no trace of the active site could be detected. It is thought that something, most likely oxygen contamination, about the process of transferring the protein from the glove box to the spectrometer at

Mines that disrupts the proteins.

47 Figure 3.15: Spectra of CaI in various stages of drying; (a) 20mg/mL CaI in Tris buffer; (b) 18mg/mL CaI in Tris buffer (no glycerol); (c) CaI film scanned once dry (about 1 hr) on CaF2 window and left in spectrometer for further scanning; (d) CaI dried film after about 2.5hrs; (e) CaI dried film after about 5hrs; (f) CaI film dried on separate CaF2window and

kept at 4 °C in glove box for 5.75 hrs. Green highlights correspond to typical auto-oxidized CaI modes. Peaks that appear to have shifted after films have dried are highlighted in blue. Peaks that correspond with reduced CaI are highlighted in red. The peak at 1996cm−1 is unassigned.

48 Figure 3.16: Comparison of CaI films dried (a) in a transmission cell, (b) on a horizontal ATR prism at NREL (c) on a vertical ATR prism at Mines

49 CHAPTER 4 CONCLUSION

The objectives of this thesis were to study novel methods and materials to develop a superior platform for the spectroelectrochemical study of hydrogenase enzymes. Many of the methods developed to improve upon a basic spectroelectrochemical cell were found to have their own strengths and weaknesses. The evolution of these methods were guided by the capitalization of certain advantages while overcoming the challenges of each technique. The most basic spectroelectrochemical cell developed used doped Si engineered to be conductive yet IR transparent. As an electrode, doped silicon was able to oxidize and reduce species but eventually the formation of an oxide layer diminished this ability. Growth of the oxide could be prevented by functionalization with a siloxane-based SAM, however, this still required an oxide layer for a well-ordered SAM to form on. Since an oxide layer was overly insulating, the notion to simply replace it with a Au coating was explored. By removing the native oxide before depositing evaporated Au onto a doped wafer no further oxide would form and thiol-base SAMs could be used to improve biocompatibility. The use of electrolessly deposited Au over the entire length of an ATR prisms was investigated but the number of challenges to fully develop the technique were considered, at the time, to be too many to practically overcome within this work. Inspired by the notion nanostructured Au, the use of

NPGL films were studied to see how they compared as electrodes against Au coated doped Si electrodes. It was determined that the use of NPGL films is of immense interest in the general context of spectroelectrochemistry. Exhibiting the high conductivity of bulk gold but still IR transparent, NPGL films turned out to be an excellent material as a working electrode that, as a bonus, could be used with the ZnSe prism. Eventually, the use of NPGL as a nanoporous membrane was found to be a useful technique for confining proteins in solution to a small volume while still allowing chemical interaction with a bulk electrolyte.

50 Studies with myoglobin show that the use of a nanoporous filter of any variety to confine proteins close to an ATR surface maximizes the efficiency with which proteins are used. Before actually using the latest technique to study CaI, the process of drying CaI films had to be scrutinized. Spectra of CaI dried or drying show that the process shifts peaks and gives rise to new ones not all of which can be fully explained. It is obvious that dehydrating the films changes the protein environment upon which its structure and activity are highly dependent thus changing their associated spectra but further studies should be done to more carefully quantify these effects. Once the effect drying has on hydrogenases is better understood, or at least demonstrated to not disrupt the active

site, then the use CaI films can be used in combination with all other methods. A floodgate of research opportunity would open as CaI films could be studied with NPGL films. The pores within a NPGL film etched for 35 min are large enough to allow CaI within the pores but prevent any significant flow through the film. Ideally, a NPGL film immersed in a CaI

solution for long enough would allow them to diffuse into the pores and benefit from the potential IR absorbance enhancement that appears to occur for 35 min etched NPGL while effectively remaining trapped within the pores. Further, and more diligent, research into the formation of an electrolessly deposited Au

film over a large area would be required in order to obtain a multi-bounce SEIRA setup. The successful formation of a uniformly islanded Au film would allow for an order of magnitude enhancement of absorbance due to multiple bounces in addition to the two to three order of magnitude enhancement due to nanostructured Au. This would allow allow for the study of only hydrogenases bound to the surface and would not need a concentrated film near the

surface to provide any significant spectral data. Ultimately, a continuing process of evolving and combining these techniques would be beneficial both to enhance the capabilities of CSM and the field of spectroelectrochemistry in general.

“If we knew what it was we were doing, it would not be called research, would it?” - Albert Einstein

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56 APPENDIX - ALTERNATIVE METHODS AND MATERIALS EXPLORED

A.1 CO inhibition of Myoglobin

The spectra of CO inhibited Myoglobin were taken for most experimental configurations as a method of comparison. Changes in vibrational modes of the heme group in myoglobin due to dehydration have already been extensively characterized and are spectroscopically simple [45]. Dehydrated myoglobin have discrete shifts of the FeCO mode from 1945cm−1 to 1967 cm−1. Myoglobin films used in this work were often only partially dehydrated resulting in a more broad composite peak shown in Figure A.1. The data shown in Table A.1 uses the

Figure A.1: Partially hydrated Mb film with composite spectrum in blue, hydrated film component in purple and dehydrated film component in yellow. total mass of protein in a film. Since partially dried films would have more absorbance modes split between 1945 cm−1 and 1967 cm−1the total area between 1920 cm−1 and 1980 cm−1 is also used for comparison. The peak improvements are calculated by dividing the peak efficiency of each sample (Area/mg or Height/mg) by the the peak efficiency of a film with no cover. Certain methods were with the doped Si ATR and is provided for comparison in Table A.2.

57 Table A.1: Comparison of CO-Mb films drop cast from DI water (with the exception of method a) on a ZnSe ATR in various configurations: (a) drop cast from Tris buffer with 5% glycerol covered with paper; (b) covered with 15 min etched NPGL on paper; (c) covered with 15 min etched NPGL on paper; (d) covered with 35 min etched NPGL on paper; (e) covered with NPGL 35 min etched NPGL; (f) covered with NPGL 35 min etched NPGL; (g) drop cast on MPA functionalized NPGL adhered via MPS to Si wafer optically coupled to ZnSe ATR; (h) drop cast on TESBA functionalized Si wafer optically coupled to ZnSe ATR; (i) drop cast on Si wafer optically coupled to ZnSe and covered with paper; (j) no cover

Peak data Peak Improvement Method Mass (mg) Area Height Area/mg Height/mg Area Height a 0.1 0.0548 0.0017 0.548 0.0174 3.28 2.90 b 1.6 0.1149 0.1230 0.072 0.0769 0.43 12.81 c 2.0 0.3372 0.1230 0.169 0.0615 1.01 10.25 d 0.1 0.0158 0.0005 0.158 0.0052 0.95 0.87 e 1.0 0.2406 0.0079 0.241 0.0079 1.44 1.32 f 0.1 0.0234 0.0008 0.234 0.0075 1.40 1.25 g 0.1 0.0150 0.0005 0.150 0.0054 0.90 0.90 h 0.1 0.0022 0.00008 0.022 0.0008 0.13 0.13 i 2.0 0.0256 0.00084 0.013 0.0004 0.08 0.07 j 1.0 0.0143 0.0005 0.0143 0.0005 1 1

Table A.2: Comparison of CO-Mb films drop cast from DI water on a doped Si ATR in varying configurations: (a) drop cast on top of one day etched NPGL; (b) drop cast on top of 35 min NPGL adhered via MPS; (c) covered with paper; (d) no cover

Peak data Peak Improvement Expt Mass (mg) Area Height Area/mg Height/mg Area Height a 0.1 0.0059 0.00023 0.059 0.0023 4.11 4.51 b 0.1 0.0018 0.00007 0.018 0.0007 1.22 1.37 c 2.0 0.0157 0.00085 0.008 0.0004 0.55 0.83 d 1 0.0143 0.00051 0.0143 0.0005 1 1

58 It is evident that using a cover confines enough of the protein film close enough to the surface to obtain stronger spectra with less protein than without a cover. Although pore sizes of the NPGL are large enough to allow proteins through the overall diffusion is very slow compared to the time frame of most experiments. The use of paper and/or NPGL also acts as a flow sheer barrier such that solutions can be injected while the cover prevents loosely bound proteins from being swept away. It is unclear whether the flow barrier or confinement properties of a cover contribute more to observed spectroscopic absorbance enhancement.

A.2 Electroless Gold

After a review of the literature it was noted that procedures for creating an electrolessly deposited Au film for SEIRAS were similar but differed in some key ways. All procedure require the a silicon substrate to be immersed in NH4F for 1 − 3 min to terminate all silicon surface sites with hydrogen. All papers used a plating solution which composed of 0.015M

NaAuCl4 + 0.15M Na2SO3 + 0.15M Na2S2O3 + 0.05 M NH4Cl and mixed this in a 2:1 ratio with 2% HF before dropping the solution on freshly hydrogen terminated Si. After an amount of time the deposition reaction would be quenched by washing the plating solution away with DI water. In a paper by Miyake et al. [46], the final mixture is dropped onto

the Si surface for 60 − 90s at 60 °C. In a paper by Jin et al. [47], HAuCl4is used instead

of NaAuCl4in the same concentration but the solution is deposited at 60 °C for 3 min in the dark. In a paper by Ataka and Heberle [48], the plating mixture is listed as a 1:1:1 mixture

of 0.03 M NaAuCl4: 0.3M Na2SO3 + 0.3M Na2S2O3 + 0.1 M NH4Cl: 2% HF respectively, which results in the same final concentrations as listed by the other papers indicating that

perhaps the mixing of Na2SO3, Na2S2O3 and NH4Cl together before the addition NaAuCl4 was somehow relevant. The temperature of the deposition was not specified but was said to last 60 − 90 s. Some procedures also specify that the Si prism deposition was polished with either 0.3 µm or 0.03 µm alumina powder and it is unclear whether the this affects the Au deposition. The primary characteristic of a gold layer capable of enhancing infrared absorbance is an assortment of closely packed Au islands with an average diameter of about

59 300 nm and average film thickness of 60 nmfor procedures requiring only 60−90 s depositions [46, 48]. An average particle size of about 100 nm, however, is reported for procedures with

HAuCl4for 3 min in the dark [47]and still have significant absorbance enhancement. It is unclear whether the plating solution were also heated or if they were deposited at room temperature. Initially problems were encountered where Au particles would precipitate out of solution while mixing the plating solution to make black ink-like or brown solutions. It is thought this was due to minute concentrations of contaminants, thus, any and all labware to came in contact with the plating solution was cleaned with piranha solution for ˜10 min then with a heated 6:1:1 mixture of DI water, Concentrated HCl, 30% H2O2 respectively for about 10 min. With extremely clean labware the plating solution would usually remain completely transparent. Large black grains would tend to form if the solution was left for more than a few hours so fresh plating solution was made for each deposition. After each deposition the average thickness was measure with profilometry and the sheet resistance was measured with a 4-point probe. Films with sheet resistances above about 1000 Ω/sq were not considered for further testing. In many cases portions of the deposited Au would be washed away either during quenching or while rinsing the sample with DI water. Samples were portions of the

films had been washed away were also not used for further testing.

A.2.1 Small area depositions

Au was deposited by heating a single side polished wafer on a hot plate to 60°C then

making a drop ≈ 1 cm in diameter of plating solution. Solution was allowed to deposit Au for 30 − 90 s before quenching the reaction with DI water. Robust, conductive Au only occurred in ring shaped regions at specific distances from the center of the drop. If a deposition was allowed to take longer than about 90 seconds the gold would start to appear rough rather than shiny and these regions would wash away with water. The outer regions of a drop allowed to deposit gold for 30 sec adhered to the silicon after rubbing the surface with a q-tip while only the inner regions of a drop allowed to deposit gold for 60 sec adhered.

60 Depictions of electroless Au deposition show a hemispherical or triangular prism that is set in a water bath with plating solutions generously covering the entire prism so it was thought these differences created the discrepancies between published results and those obtained in the lab.

The Au layers would go through three different phases. In the initial phase the Au gradually deposits but appears dull. In the second phase the Au becomes more lustrous and appears similar to bulk Au. If the deposition is allowed to continue to the third phase the Au starts to appear as though a layer of frost was forming on the surface. Typically any portion of the Au that reaches this third phase is instantly washed away by the water used to quench the reaction. When examined under a microscope Au that in the third phase has cracks that appear similar to dried mud. If a Au film is still in the first phase at the end of a deposition it will be substantially less conductive and would make for a poor electrode. Thus the issue with creating a large area gold film is ensuring the entire film in this “Goldilocks” phase. Any slight variation in temperature across the length of the wafer appears to create a non-uniform gold layer.

A.2.2 Large area depositions

This method was to be an adaption from Ataka and Heberle [48] for large area wafers, ideally to combine the benefit of surface enhanced infrared absorption spectroscopy with a large sample area. The degree to which all portions of a single wafer must be the same necessitated several iterations of a proper wafer/water bath/plating solution configuration.

The final configuration is shown in Figure A.2. The purpose of the primary water bath is to contain the plating solution once it is washed away. The metal block provides an isothermal flat surface kept in equilibrium with the secondary water bath. The glass slide between the wafer and the metal block is slightly smaller than the wafer so that no plating solution leaks onto the metal block until after

the wafer is gently dowsed with DI that was also 60°C. The water baths and metal block

were allowed to thermally equilibrate for several hours at 60°C. The wafers used in the final

61 Figure A.2: Double water bath for thermal isotropy during electroless Au deposition. depositions were double-side polished p-type wafers about slightly longer and wider than a standard 1 inx3 in glass slide. One of the final Au films produced had 6 ml of plating solution dropped as quickly and uniformly as possible and allowed to react for ≈ 55 s. The Au layer had uniform conductance with sheet resistances between 8 and 13 ohms/sq across the entire surface. To test the Au films produced for any enhanced absorbance they were assembled in the ZnSe/coupling fluid-wafer-flow cell stack. Ethanol was injected in the flow cell and scanned at a background follow by a solution of 2% v/v MPA in ethanol. The coupling fluid used in our assembly has an incredibly strong signal: even the slightest change in the coupling fluid dramatically changes its absorbance spectrum which gives it a large signal in any difference spectra. The change in absorbance is not the same for all regions of the spectrum making background subtraction exceptionally difficult. (NOTE: I don’t have a data set of evaporated

Au film with all the same control variables to compare against the electroless Au. Going back through the data there was always some variation that makes it impossible to assign effects to specific causes and in general my methods for determining whether the electroless gold was enhancing absorbance of surface species were rather flawed. There’s not really a complete and logical story I can tell with the IR spectrum. I did notice that all the AFM scans of the electroless Au actually didn’t have proper islanding so I can say that we stopped

62 using electroless gold for that reason which is indirectly true.) Multitudes of electrolessly deposited Au films were created and imaged with AFM but for the sake of simplicity only two are analyzed for this section. Figure A.3 shows the typical nanostructure of electroless gold deposited for ≈ 60 susing a plating solution with HAuCl4. Though there are island like features the average diameter is only 82 nm (as determined by Nanoscope 5.3 software analysis) and rarely is an island larger than 200 nm.

Figure A.3: AFM images of electroless Au attempt

The next most reasonable experiment would be to test a plating solution with HAuCl4with a 3 min deposition time, however, for reasons since forgotten, a 3 min deposition using a plat-

ing solution with NaAuCl4was studied instead. The film, shown in Figure A.4, has larger islands where the average diameter is 175 nm. Unfortunately, it would appear that even if islands of the desired size could be obtained through even longer depositions, the density

63 of islands would be too great for them to be distinguished from one another. Thus, to cre- ate electrolessly deposited Au films with the same island-like structure seen in Au films for SEIRAS the initial density of nucleation sites would need to be much less.

Figure A.4: AFM image of 3 min Au

It is possible that the acidity along with slight differences in deposition conditions greatly affects the deposition process and after further review of experimental procedures used during this time period indicate that SEIRAS may have even been successful but a lack thorough and repeatable testing prevented conclusive results from being obtained. Even if a film had proper island sizes it may not have been used if it was wiped off too easily, a condition for success that in retrospect was not necessary. Also, the procedure for determining if an electrolessly deposited film could be used for SEIRAS was to functionalized the films in situ with MPA and see how much stronger the MPA spectra was after about an hour, however,

64 an ex situ deposition of MPA for about twenty hours would have provided more conclusive results.

A.3 Evaporated Au

The creation of evaporated Au thin films was initially necessary for testing the electro- chemical capabilities of the flow cell. In preliminary experiments, Au films were tested in a large volume (100 mL)electrochemical cell where a lengthy process of using epoxy and sil- ver paint was used to turn the films into compatible electrodes. Since the majority of Au

films would peel off from the substrate due to the admittedly extreme potentials applied, a quicker testing solution was devised. Figure A.5 shows what is called in this work the minicell which can be clamped directly onto the surface of an electrode and immediately used for testing. Typically a solution of 0.5 M H2SO4or 10 mM Fe(CN)6 in PBS was injected into the minicell to test a working electrode. Although the volume is somewhat small the cell is not strained by the thin-film geometry of the small volume flow cell and thus behaved like a typical electrochemical cell.

The cyclic voltammogram of thick Au electrodes with 0.5 M H2SO4 was taken in both the minicell and the small volume flow cell as shown for comparison in Figure A.6. The large shift in observed potential is to be expected and the small oscillations seen the flow cell CV where not deemed to be problematic. The process of cycling voltage with a sulfuric acid electrolyte is typically used to clean Au surfaces. In AFM scans, shown in Figure A.7, bumps are seen on the surface however it is unclear if these are contaminants or non-uniformities of the Au layer. After electrochemically cleaning the Au films the bumps appear to have been removed leaving flat, clean Au. Once the Au films had been proven to be electrochemically stable, thin Au coatings over doped Si were investigated. Due to the complexity and generally unexplainable results obtained from a large number of electrochemical test comparing thick Au films to doped Si and Au coated doped Si not many conclusions could be made other than the fact that

65 Figure A.5: Minicell setup

Figure A.6: CV of 0.5 M H2SO4 with thick Au working electrode using the small volume flow cell (red), and the minicell (blue)

66 Figure A.7: 20 nm evaporated Au film on Si after rinsing with DI and acetone (left). Elec- trochemically cleaned 20 nmevaporated Au film (right) an analyte could be repeatably oxidized and reduced. The best results were the obtained using buffers with pH further from neutral which improved ionic conductivity as illustrated in Figure A.8.

Figure A.8: Cyclic voltammograms of ferricyanide using MPA functionalized Au coated doped Si using pH 4 PBS (left) and pH 7 PBS (right).

67 A.4 Doped Si fabrication and characterization details

The process of finding a concentration and thickness of a doped layer sufficient for elec- trochemical studies while still remaining transparent in the IR was necessary to create doped

Si wafers or prisms that could be used in a spectroelectrochemical cell. (NOTE: some of the information I used when making these wafers, such as the typical diffusion depths, were things experimentally determined the entire class during the Microelectronics Processing Lab with Dr. Collins, should I elaborate on this?).When wafer samples were doped the initial sheet resistance (about 3.37 Ω/sq) corresponded to a diffusion depth of only 4.3 µm which are usually obtained from a thirty minute deposition at lower temperatures. The higher than expected sheet resistances indicate the diffusion oven did not have a sufficient time to heat

to 1250 °C. Wafers were etched in∼ 0.5 µm increments in a March Instruments CS 1701

Reactive Ion Etcher (RIE) at 250 mTorr with a constant SF6 flow of 48 sccm and RF power of 200 W. Sheet resistance and transmission spectra were measured between etches. Etch depth was verified with profilometry. The change in transmission spectra (Figure A.9) are a result of shifting the plasma frequency away from the infrared.

−1 Beyond about 4 ñm etch depth the wafer is completely transparent above 650 cm .

Beyond 4.5 − 4.8 µm (past the expected dopant diffusion depth) the wafer ceased to be conductive enough to serve as a working electrode. I was able to check this against resistivity data to verify how accurate my model was. Results indicate my actual diffusion depth of the spin-on dopant was about 4.5 µm and etching down to 4.2 µm provides a thin doped layer

with carrier concentration of about 2.7 ∗ 1019cm3.

A.5 Carboxyethylsilanetriol Monolayer Fabrication and Characterization

While many procedures for obtaining CSS monolayers on oxide free silicon require lengthy procedures involving reactions with argon radicals Pinson [39] recent advances in biosensors have shown that the formation of CSS monolayers sufficient for protein immobilization can

be achieved with simple aqueous depositions Duval et al. [49]. Attempts to quantify the

68 Figure A.9: Successive Etching of Highly Doped Si Wafers. Perturbations in spectra are due to variances in CO2and water vapor.

69 quality of CSS monolayers made by these aqueous depositions were made using a variety of methods. Initially, the temperature of the deposition solution and the effects of annealing were tested on double side polished p-type Si wafers cut to 1 in squares. All wafers were cleaned in a UV-Ozone Cleaner for 15 min prior to deposition. Two wafers were immersed

for 40 min in acidic solutions of 1% v/v CSS in DI water at room temperature (∼ 20°C)

and two others were immersed in identical solution heated to 90°C. Wafers were rinsed with DI water and dried under nitrogen. One wafer from both the room temperature (RT) and

high temperature (HT) were annealed at 80°C. To characterize the monolayer sessile water contact angle measurements were taken using a Logitech Orbit AF webcam and processed

with drop-snake in ImageJ. Both acidic and basic drops were used to demonstrate the change in surface energy from titration of carboxyl groups with hydrogen chloride (pH 3) and sodium hydroxide (pH 13). Prior to contact angle measurements all wafers were immersed in pH 3 HCl solution to make sure all carboxyl groups were protonated. For all basic drops the

contact angle was too low to be processed reliably in drop-snake. Acidic drops had high enough water contact angles to show variations depending on CSS deposition procedure as shown in Figure A.10. The average water contact angles of drops on annealed surfaces were higher than for those on non-annealled surfaces. There was no significant difference between

high temperature depositions and room temperature depositions when the samples were not annealed. There was a significant increase in contact angle when CSS was deposited at high temperature and then annealed, however, this sample also had the highest standard deviation. The pH during deposition also plays a role in the quality of CSS monolayers since solu- tions above about pH 9 will cause CSS dimers to form Aureau et al. [50]. For experiments with varying pH depositions, in addition to UV-Ozone cleaning for 15 min, all wafers were immersed in a buffered oxide etch (BOE) for one minute. The native oxide thicknesses were measure with ellipsometry after wafers were removed from the BOE. Wafers were immersed in a room temperature solution of 1% v/v CSS with varying pH for 40 min. Water contact

70 Figure A.10: Sessile water contact angle measurements for temperature dependent deposition of CSS monolayers

angles and ellipsometry measurements were taken after rinsing wafers with DI water and drying with nitrogen. Ellipsometry measurements for low pH depositions tend to follow the

same trend as water contact angles. After a deposition at pH 11.66 (unadjusted CSS solu- tion) the thickness is dramatically increased and darker regions are visible on the surface of the wafer. Figure A.11 shows depositions in acidic solutions generally result in greater contact angles but the difference between acidic and basic water contact angles remain relatively similar. It is unclear if this means more carboxyl groups are on the surface or if the monolayer is more ordered. The surface topology of these wafers was imaged by AFM for all samples deposited in acidic solutions. Samples immersed in CSS solutions with pH 6.5 appeared to form aggregates. Wafers immersed in CSS solutions with pH 5 start to develop circular craters of similar depth which are likely areas where CSS did not deposit. Between pH 2 and pH 4 uniform layers of CSS appear to have formed. At pH 1 the surface appears rough indicating a non-ordered monolayer or multilayer of CSS was formed. Figure A.12(a)Fig- ure A.12(b)Figure A.12(c)Figure A.12(d)Figure A.12(e)

71 Figure A.11: Sessile water contact angles of acidic and basic drops on CSS monolayers de- posited with different pH levels and corresponding thicknesses as determined by ellipsometry

IR spectra ( Figure A.13)of an in situ formation and titration of CSS monolayers were obtained. A Si ATR prism was rinsed with acetone then UV-Ozone cleaned for 15 min prior to use. Using a small volume flow cell 0.1 M HCl was used as a background then 1% v/v

CSS in 0.1 MHCl was injected and left to deposit for 40 min. The flow cell was flushed with DI water then injected with 0.1 MNaOH with spectra obtained after each injection. The formation of a CSS layer of some kind has taken place indicated by the large car- boxyl and carboxylate peaks at 1548 cm−1 and 1768 cm−1 respectively which similar but not identical to results in the literature Aureau et al. [50]. Smaller peaks at 2849cm−1,

2918 cm−1 and 2963 cm−1 also appear corresponding to short alkyl chains Tian et al. [51]. The peak at 1655 cm−1 is assumed to be due to water. Assuming an ordered CSS mono- layer was formed then after injecting 0.1M NaOH the carboxyl peak at1768cm−1 should have decreased while the peak at 1768 cm−1 increased proportionally. Instead the carboxyl peak increases slightly, a new broad peak at 2068 cm−1 appears and the akyl peaks between 2800 cm−1 and 3000 cm−1 appear to shift slightly. Ellipsometry measurements of the ATR crystal indicated a change in thickness of about 6-7A˚ which is consistent with the thick- nesses of a monolayer deposited at room temperature with no annealing (about 5A).˚ These

72 (a) CSS deposition at pH 1 (b) CSS deposition at pH 2

(c) CSS deposition at pH 3 (d) CSS deposition at pH 4

(e) CSS deposition at pH 5

Figure A.12: AFM images with 512 samples at 1.001 Hz from Si wafer functionalized with CSS at varying pH levels.

73 Figure A.13: CSS monolayers formed on Si ATR surface and subsequent deprotonation with NaOH.

74 results indicate that deposition of CSS solutions between pH 1 and pH 4 likely produce CSS monolayers. It is unclear how ordered these monolayer are, however, it should be noted that other papers appear to functionalize silicon surfaces sufficient enough to couple with protein films Duval et al. [49].

A.6 Meltmount

The use Cargille Meltmount, a high index thermoplastic, was hypothesized to be a more stable coupling medium between the ZnSe ATR prism and a Si wafer. Since Meltmount is not volatile there is no maximum time limit that it could be used in an optically coupled setup as is the case with diiodomethane. Figure A.14 shows that even though the Meltmount is not evaporating it is certainly changing over time. This is problematic because the Meltmount changes differently over every time interval scanned. Figure A.14 shows only six scans over one hour but the data continues to have non-recurring changes in spectrum. Although rapid electrochemical modulation can be used to obtain an oxidized-minus-reduced spectrum, the results tend to have excessive noise and deemed unusable for use with hydrogenases.

A.7 NPGL enhancement studies

The use of NPGL film has the potential to become a branch of science in its own right and the variety of ways it can be used for the spectroelectrochemical study of proteins done in this work only scratches the surface. NPGL films were made with different etch times to see if there was an etch time that resulted in signal enhancement of adsorbed species. Each film was transferred to a ZnSe prism and allowed to dry before immersing in a solution of 1% MPA in ethanol for 19.5 hrs. Each sample scanned was before and after functionalization with the former serving as a background for the latter. The results shown in Figure A.15 indicate that around for etch times around 35 min a clear increase in peak intensity at 1735 cm−1 occurs. Comparing these results to known surface area enhancements shown in Figure A.16 for various etch times it becomes apparent that the peak intensities at1735 cm−1 do not follow

75 Figure A.14: Difference spectra between scans taken every ten minutes

76 Figure A.15: 19.5 hr MPA deposition on NPGL film etched for various times: (a) 15 min; (b) 35 min; (c) 3 hrs; (d) 1 day

77 the same trend. Thus the large enhancement for 35 min etched NPGL films is likely not due to increased surface area but some other property of the film.

Figure A.16: Surface area enhancement of NPGL etched for various times. Adapted with permission from Ciesielski et al. [37]. Copyright 2008 American Chemical Society

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