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Electrochemical study of proteins using electrodes with modified surfaces By Hamid Fini A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Chemistry University of Toronto © Copyright by Hamid Fini 2020

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Page 1: Electrochemical study of proteins using electrodes with ......ii Electrochemical study of proteins using electrodes with modified surfaces, Hamid Fini, Doctor of Philosophy, Chemistry,

Electrochemical study of proteins using

electrodes with modified surfaces

By

Hamid Fini

A thesis submitted in conformity with the requirements for the

degree of Doctor of Philosophy

Department of Chemistry

University of Toronto

© Copyright by Hamid Fini 2020

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Electrochemical study of proteins using electrodes with modified surfaces, Hamid Fini,

Doctor of Philosophy, Chemistry, University of Toronto, 2020

Abstract

Some different aspects of proteins properties as well as nanostructures were studied using

various electrochemical approaches in this thesis. Electrochemical impedance

spectroscopy (EIS), cyclic voltammetry (CV), differential pulse voltammetry (DPV) were

employed as well as many other analytical tools and methods to explore some structures

of our interest. In the first chapter, the above-mentioned electrochemical methods along

with some fundamental background of electrochemistry were reviewed. The chapter, also,

reviews some aspects of membrane proteins, membrane double layers and chemistry of

diazonium compounds which were employed in our studies. The structure of a novel (and

patented) nanostructure and mainly its response to electrochemical impedance

spectroscopy was studied and discussed in chapter 2. We showed how we can isolate the

response of this nanosensor to a small change of the dielectric constant of the material

entraps in the nanogap of the sensor. In chapter 3, we used DPV and CV methods to

investigate nitrite reduction activity of human hemoglobin. We proposed -for the first

time- a complete reaction mechanism for the electrochemical reactions taking place on

the surface of modified glassy carbon electrodes. We showed how these electrochemical

results were comparable to spectroscopic methods and can be used for future studies of

hemoglobin nitrite reduction studies. In chapter 4, we developed a novel platform for

preparing a hybrid bilayer (HBM) for electrochemical studies of membrane proteins. We

used diazonium chemistry to graft the first layer of the HBM covalently to the glassy

carbon electrode. We demonstrated the flexibility of our platform for developing various

bilayers with relatively predictable characteristics using molecules with desired

properties as the first and second layer in HBM. We used two different electrochemical

probes with opposite charges to show how differently the HBM responded to these probes

depending of the charge of the polar groups on the second layer. We used amyloid-β42 to

demonstrate the application of this platform to study protein aggregation processes. In

chapter 5, we discussed some future directions for all previously discussed chapters and

mentioned some potential studies which may follow these studies.

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Acknowledgements

It is priceless to have a great supervisor as a graduate student. I appreciate Professor

Kagan Kerman not only for all his supports, supervision and scientific hints and helps,

but also for his peaceful mind and patience which helped me live my PhD years. He gave

me another life like my parents did for the first time. I shall never forget his greatness.

I had an absolute honor to have Professor Gilbert Walker, Professor Cynthia Goh as my

supervisory committee members. Their helps and support were invaluable for me. I would

like to take the opportunity to thank my exam committee members Professor Aaron

Wheeler, and Professor … for offering their precious time and helpful comments to

improve the quality of my thesis. I also want thank Professor Al-Amin Dhirani that I had

a chance to work in his lab for a period of time. He taught me how to think scientifically.

I had a great opportunity to work with many great people in University of Toronto; I want

to appreciate all of them, so Thank you UofT. My special thanks are for Anna Liza

Villavelez who supported me and helped me to find my way to Professor Kerman’s group.

Finally, my life was so nice having dear friends and colleagues at UofT, specially my

friend in Professor Kerman’s group. Qusai Hasan, Hashwin Ganesh, Jacky Li, Han Su,

Soha Ahmadi and many others made my life more beautiful at UofT campus.

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Table of Contents

Abstract……………………………………………………………………………….ii

Acknowledgements..…………………………………………………………….…...iii

List of Tables………………………………………………………………......……….

List of Schemes……………………………………………………...………………...

List of Figures………………………………………………………………...………...

List of Appendices……………………………………………………………...…….

List of Abbreviations………………………………………………………...……….

Chapter 1 ........................................................................................................................... 1

1. An introduction to electrochemical sensors and biosensors .............................. 1

1.1 Electrochemical biosensors .................................................................................. 1

1.2 An introduction to electrochemical techniques .................................................... 5

1.2.1 Electrical charge, current and voltage ......................................................... 5

1.2.2 Electrical elements ........................................................................................ 6

1.2.3 Direct current (DC) voltage and alternating current (AC) voltage ............. 7

1.2.4 Electrical impedance .................................................................................... 7

1.2.5 Impedance spectroscopy ............................................................................... 8

1.2.6 Electrochemical cell and impedance spectroscopy: constant phase element,

Warburg element, and Randles equivalent circuit .................................................... 15

1.2.7 Electrochemical Cell ................................................................................... 18

1.2.8 Faradaic and non-Faradaic processes ....................................................... 18

1.2.9 Electrical double layer capacitance and voltage step ................................ 20

1.2.10 Controlled potential experiments: Linear sweep voltammetry, cyclic

voltammetry and differential pulse voltammetry ....................................................... 23

1.2.11 Multicomponent systems and multi-step charge transfer ........................... 31

1.2.12 Effect of pH on peak potentials ................................................................... 32

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1.2.13 Calculation of number of electrons for an electrochemical reaction using

CV and DPV .............................................................................................................. 32

1.3 An introduction to Bilayer and Surfactant membranes and chemistry of

diazonium salts used as electrode surface modification in electrochemical sensors ... 35

1.3.1 Bilayer membranes ..................................................................................... 35

1.3.2 Surfactant membrane and films .................................................................. 36

1.3.3 Diazonium salt chemistry ............................................................................ 39

1.4 Overview of next chapters .................................................................................. 42

Chapter 2 ......................................................................................................................... 43

2. Microfabricated, silicon devices with nanowells and nanogap electrodes: a

platform for dielectric spectroscopy with silane-tunable response .............. 43

2.1 Connecting text ................................................................................................... 43

2.2 Abstract ............................................................................................................... 45

2.3 Introduction ........................................................................................................ 46

2.4 Experimental section .......................................................................................... 48

2.4.1 Impedance spectroscopy instrumentation. .................................................. 48

2.4.2 Nanogap device cell compartment. ............................................................. 48

2.4.3 SEM imaging ............................................................................................... 48

2.4.4 Etching procedure ....................................................................................... 49

2.4.5 Silanization procedure ................................................................................ 49

2.5 Results and discussion ........................................................................................ 50

2.5.1 Nanogap device structure ........................................................................... 50

2.5.2 Impedance spectroscopy instrumentation ................................................... 51

2.5.3 Frequency response and equivalent circuit for the nanogap device .......... 54

2.5.4 Sensitivity of the nanogap device to the dielectric constants of various

media 58

2.5.5 Surface sensitivity of Nanogaps .................................................................. 62

2.5.6 Probing nanogaps by means of double layer thickness .............................. 63

2.6 Conclusion .......................................................................................................... 64

2.7 Acknowledgments ............................................................................................... 64

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Chapter 3 ......................................................................................................................... 65

3. Revisiting the nitrite reductase activity of hemoglobin with differential pulse

voltammetry ...................................................................................................... 65

3.1 Connecting text ................................................................................................... 65

3.2 Abstract ............................................................................................................... 67

3.3 Introduction ........................................................................................................ 67

3.4 Experimental ....................................................................................................... 70

3.4.1 Materials ..................................................................................................... 70

3.4.2 Instruments .................................................................................................. 70

3.4.3 Electrode modification ................................................................................ 70

3.4.4 TEM............................................................................................................. 71

3.4.5 Electrochemical analysis ............................................................................ 71

3.5 Results and discussion ........................................................................................ 72

3.6 Conclusion .......................................................................................................... 86

3.7 Acknowledgments ............................................................................................... 86

3.8 Supplementary information ................................................................................ 87

3.8.1 Discussions about the capacitive current observed in cyclic voltammograms

87

Chapter 4 ......................................................................................................................... 90

4. A novel hybrid bilayer membrane as a platform for electrochemical

investigations of membrane proteins .............................................................. 90

4.1 Connecting text ................................................................................................... 90

4.2 ABSTRACT ......................................................................................................... 92

4.3 INTRODUCTION ............................................................................................... 92

4.4 EXPERIMENTAL METHODS ............................................................................ 94

4.4.1 Chemicals .................................................................................................... 94

4.4.2 Instruments .................................................................................................. 94

4.4.3 Electrode pretreatment ............................................................................... 95

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4.4.4 Synthesis of 4-dodecylbenzenediazonium tetrafluoroborate (DDAN) ........ 95

4.4.5 Synthesis of EDAN ...................................................................................... 96

4.4.6 Electrode modification ................................................................................ 96

4.4.7 Electrochemical measurements .................................................................. 97

4.4.8 Aβ1-42 aggregation studies ........................................................................... 97

4.5 RESULTS AND DISCUSSION ........................................................................... 97

4.5.1 Covalent modification of GCE surfaces ..................................................... 97

4.5.2 Preparation of the hybrid bilayer membranes (HBMs) ............................ 100

4.5.3 Electrochemistry of lcHBM- and scHBM-modified surfaces .................... 104

4.5.4 Interaction with Aβ1-42............................................................................... 107

4.6 CONCLUSIONS ............................................................................................... 111

4.7 ACKNOWLEDGMENTS................................................................................... 111

4.8 Supplementary information .............................................................................. 112

Chapter 5 ....................................................................................................................... 119

5. Future directions ................................................................................................ 119

5.1 Microfabricated, silicon devices with nanowells and nanogap electrodes ...... 119

5.2 Nitrite reduction activity of different types of hemoglobin ............................... 124

5.3 Covalently attached spread bilayer on glassy carbon electrode using diazonium

salts 125

References ...................................................................................................................... 126

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List of Tables

Table 1.1 Methods used for calculation of number of electrons for known electrochemical

reactions ............................................................................................................................ 33

Table 1.2 Calculated number of electrons for known electrochemical reaction using

methods in Table 1.1 ......................................................................................................... 34

Table 2. 1 Equivalent circuit model parameters for the nanogap device in air and

propanol. ........................................................................................................................... 56

Table 3. 1 The calculated number of electrons and protons that were exchanged in the

redox processes at electrode surface (depicted as redox couples A, B and C). ................ 75

Table 3S. 1 Peak potentials (Ep) of Hb-DDAB-modified GCEs under various pH

conditions. Each potential value is the average of five different scans. The solution was

purged by nitrogen and stirred between each experiment. All solutions contained 8 mM

nitrite. Standard deviations (SD) were calculated from five independent measurements

(n=5). ................................................................................................................................. 88

Table 4. 1 Contact angles for the bare GCE along with the contact angles for the GCE

with the ethyl and dodecyl monolayer and bilayer. ........... Error! Bookmark not defined.

Table 4S. 1 The values of simulated equivalent circuit elements of Nyquist plots shown

in Figures 6B and 6D. ..................................................................................................... 117

Table 4S.2 The values of simulated equivalent circuit elements of Nyquist plots shown in

Figure 7 with the redox probe [Ru(NH3)6]3+. .................................................................. 118

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Table 4S.3 The values of simulated equivalent circuit elements of Nyquist plots shown in

Figure 8. .......................................................................................................................... 118

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List of Schemes

Scheme 4. 1 Reaction mechanism proposed for electrochemical reaction of couple C ... 80

Scheme 4. 2 Reaction mechanism proposed for reactions occur during electrochemical

reactions couple B ............................................................................................................. 81

Scheme 4. 3 Reaction mechanism proposed for reactions occur during electrochemical

reactions couple A............................................................................................................. 83

Scheme 4. 4 Possible reactions proposed for couple D .................................................... 84

Scheme 4S.1 Schematic diagram for synthesis of 4-dodecylbenzenediazonium

tetrafluoroborate (DDAN). ............................................................................................. 112

Scheme 4S.2 Schematic diagram for in situ synthesis of 4-ethylbenzene diazonium

(EDAN). As described in the Experimental section, 2 mM of ethyl aniline was dissolved

in 1.25 M HCl followed by the addition of 1 equivalent of NaNO2. .............................. 112

Scheme 5. 1 Synthesis of CE .......................................................................................... 120

Scheme 5. 2 Modifying quartz slides using (3-Aminopropyl)methyldichlorosilane (A)

and grafted CE using aldol condensation (B) ................................................................. 122

Scheme 5. 3 Chemical structure of 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine

which is commercially available from Sigma company ................................................. 125

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List of Figures

Figure 1.1 Biocomponent and transducers used in biosensors ........................................... 1

Figure 1. 2 Ampertometirc response of a biosensor to glucose (1 mmol L-1) followed by

four injections of ATP (20 nmol L-1) measured in phosphate buffer at 650 mV in

reference to Ag/AgCl (A). Potentiometric detection of the hybridization with

complementary oligo(dT)15 on a PVC membrane functionalized with cholesterol-

oligo(dA)15 (B). A nanowire used as a conductometric biosensor (C) and its response to

the titration of complementary target DNA (red) and noncomplementary probe DNA

(blue) (n = 4 each) (D). ....................................................................................................... 3

Figure 1. 3 Cyclic voltammograms a cholesterol oxidase (ChOx) /Prussian blue (PB) sol-

gel modified GCE (B) in a phosphate buffer (pH 6.8)_0.1 mol/L KCl_0.8% Triton X-100

for a blank solution(a) and solution with cholesterol 10 (b), 20 (c), 30 (d), 40 (e), 50 (f)

nM.at Scan rate 50mV/s (A). DPV curves of an electrochemical biosensor as the

concentration of miRNA-21 varies from 0 (a) to 0.05 (b), 0.1 (c), 0.5 (d), 1 (e), 2 (f), 3

(g), 5 (h), 8 (i), and 10 fM (j). (B) while the inset is the calibration curve for the miRNA-

21 concentration from 0.05 to 5 fM. An illustration of the gold electrochemical

impedance biosensor coated (EIB) with Erythrocyte membrane (EM) and treated with

fibrinogen (Fib) (C) and the related Nyquist plots (D). ...................................................... 4

Figure 1. 4 Standard symbol for a resistor .......................................................................... 6

Figure 1. 5 Standard symbol for a capacitor ....................................................................... 6

Figure 1. 6 Standard symbol for an inductor ...................................................................... 7

Figure 1. 7 Schematic diagram of the circuit used for impedance spectroscopy. Vout is the

applied voltage by the lock-in amplifier, Rint is the internal resistance of the lock-in

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amplifier, EE stands for any different electrical elements which is being tested, RG is the

gain resistor, and Vin is the voltage across RG. ................................................................... 9

Figure 1. 8 A resistor as the “EE” (A). Measured real part (V’, blue) and imaginary part

(V’’, red) of voltage over the RG vs frequency of applied voltage (B). Bode plot of the

calculated magnitude (Z) and phase (ϕ) of impedance of “EE” vs frequency of applied

voltage (C). Nyquist plot of calculated imaginary (Z’’) vs real (Z’) part of impedance of

“EE” for the whole range of applied frequency (D). ........................................................ 10

Figure 1. 9 A capacitor as the “EE” (A). Measured real part (V’, blue) and imaginary part

(V’’, red) of voltage over the RG vs frequency of applied voltage (B). Bode plot of the

calculated magnitude (Z) and phase (ϕ) of impedance of “EE” vs frequency of applied

voltage (C). Nyquist plot of calculated imaginary (Z’’) vs real (Z’) part of impedance of

“EE” for the whole range of applied frequency (D). ........................................................ 11

Figure 1. 10 A resistor and a capacitor in parallel as the “EE” (A). Measured real part

(V’, blue) and imaginary part (V’’, red) of voltage over the RG vs frequency of applied

voltage (B). Bode plot of the calculated magnitude (Z) and phase (ϕ) of impedance of

“EE” vs frequency of applied voltage (C). Nyquist plot of calculated imaginary (Z’’) vs

real (Z’) part of impedance of “EE” for the whole range of applied frequency (D). ........ 12

Figure 1. 11 A series of a resistor and a capacitor in parallel with another set as the “EE”

(A). Measured real part (V’, blue) and imaginary part (V’’, red) of voltage over the RG vs

frequency of applied voltage (B). Bode plot of the calculated magnitude (Z) and phase (ϕ)

of impedance of “EE” vs frequency of applied voltage (C). Nyquist plot of calculated

imaginary (Z’’) vs real (Z’) part of impedance of “EE” for the frequencies from 100 Hz

to 100 kHz; data for lower frequencies are not shown in order to magnify the semicircle

(D). .................................................................................................................................... 14

Figure 1. 12 Schematic diagram of a typical electrochemical cell. The power supply

applies a voltage difference between the two electrodes. The voltmeter and the ammeter

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measure the voltage (potential, E) between the electrodes and the current (i) passes the

cell, respectively (A). the current-potential (i-E) curve for the electrochemical cell. ...... 17

Figure 1. 13 A typical model of electrical double layer ................................................... 19

Figure 1. 14 Two-electrode cell (A) with its equivalent circuit (B). A potential step (C)

which results in a transient current i (D). .......................................................................... 20

Figure 1. 15 Pathway of redox reactions on electrode (A) and the equivalent circuit (B).

........................................................................................................................................... 22

Figure 1. 16 A general set-up for three-electrode cell to run controlled-potential

measurements. ................................................................................................................... 23

Figure 1. 17 A potential step applied to an electrode (A), the resulted current (B) and the

concentration profile for various times (C). ...................................................................... 24

Figure 1. 18 A series of potential steps applied to a working electrode (A), the current-

time curves (B), the sampled-current voltammogram(C), a potential sweep (D), i-t-E

surface for a Nernstian reaction (E), linear sweep voltammetry across this surface (F). . 27

Figure 1. 19 Cyclic potential sweep (A), resulted cyclic voltammogram (B), pulse

superimposed staircase potential (C), and the resulted differential pulse voltammogram.

........................................................................................................................................... 28

Figure 1. 20 Theoretical cyclic voltammogram showing the effect of ψ and α. Curve 1:

ψ=0.5 and α=0.7, Curve 2: ψ=0.5 and α=0.3 (A). Curve 3: ψ=0.7 and α=0.5, Curve 1:

ψ=0.25 and α=0.5. (Note E1/2=EP/2) .................................................................................. 30

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Figure 1. 21 Calculated CVs for two reversible one-electron steps system where n2=n1=1

at 25oC. ΔEP is equal to -180mV, -90 mV, 0 mV and +180 mV ofr (A). (B), (C) and (D),

respectively. ...................................................................................................................... 31

Figure 1. 22 A symbol for constant phase element (A). Randles equivalent circuit (B). A

typical Nyquist plot for an electrochemical cell. .............................................................. 16

Figure 1. 23 The Fluid mosaic membrane model of a biological membrane structure. ... 35

Figure 1. 24 A gold electrode with a β-mercaptoethanol SAMs (A) and the formed

tethered bilayer lipid membranes using cholesterol/DOPC (phospholipids,1,2-dioleoyl-

sn-glycero-3-phosphocholine) as the second layer (B). .................................................... 36

Figure 1. 25 Schematic representation (not to scale) of the structure of surfactant proteins

SP-A, SP-B and SP-C, and their mode of interaction with surfactant monolayers and

bilayers. ............................................................................................................................. 37

Figure 1. 26 Schematic representation (not to scale) of the structure of surfactant proteins

SP-A, SP-B and SP-C, and their mode of interaction with surfactant monolayers and

bilayers .............................................................................................................................. 38

Figure 1. 27 A typical schematic for electrochemical reduction of diazonium salt on

conductive surfaces (A). A Glassy carbon disk electrode (d 5 3 mm) in ACN + 2 mM

+N2C6H4NO2 BF4- + 0.1 M NBu4BF4, (a) first and (b) second scan and (c) the same

electrode in ACN + 0.1 M NBu4BF4 (B). STM image of surface (a) H-Si (111) and (b)

modified with4-bromophenyl groups bonded to Si(111) (C). .......................................... 40

Figure 1. 28 An illustration of the multilayer formation during electrochemical grafting of

aryl radicals in solution to the aryl moieties bound to the carbon surface. The hydride

radicals produced in this mechanism can react with solvent or other aryl radicals. ......... 41

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Figure 2. 1(A) A schematic illustration, (B) a photograph, and (C) SEM image of the

nanogap device.................................................................................................................. 50

Figure 2. 2 Schematic diagram of the circuit used for impedance spectroscopy. Vout is the

applied voltage by the lock-in amplifier, Rint is the internal resistance of the lock-in

amplifier, Z stands for any component (nanogap device or R/C combinations) which is

being tested, RG is the gain resistor, and Vin is the voltage across RG. ............................. 52

Figure 2. 3 continued. Measured impedance responseof the nanogap device (symbols)

and calculated equivalent circuit response (solid lines) as a function of frequency.Vout is

50 mV (rms) for all the experiments. five different RG are used: 49.4 kΩ ( ), 10.0 kΩ ( ),

5.03 kΩ ( ), 999 Ω( ), 200 Ω( ). (a,b)Niquist plots for the device in air and propanol

(respectively) based on data for RG=10.0 kΩ. (c,d) Real parts of the VG(rms values)

versus frequency for the device in air and propanol (respectively). (e,f) Imaginary parts of

the VG(rms values) versus frequency for the device in air and propanol (respectively). . 54

Figure 2. 4 Equivalent circuit model for the nanogap device. Rs1 and C1 are attributed to

the annular nanogaps; Rs2 and C2to the bottom electrode/oxide/top electrode region; and

Rp to leakage current. ........................................................................................................ 55

Figure 2. 5 Imaginary part of the admittance (Z-1)'' versus ω, for frequencies from 2 Hz to

10 Hz, for a nanogap device in air and propanol (based on data shown in figure 2.4 C and

D for RG=10.0 KΩ). .......................................................................................................... 58

Figure 2. 6 Experimental data (symbols) and simulations (solid lines)for a nanogap

device in varioussolvents. For all the measurements the applied voltage was 10 mV (rms)

and RG was 10.0 kΩ. (a) Real and (b) imaginary parts of the voltage (rms values) versus

frequency. For clarity, the various plots are offset by 1.5 mV.DMSO and DEEther stand

for Dimethyl sulfoxide and Diethyl ether, respectively. ................................................... 59

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Figure 2. 7 Slope of the imaginary part of the admittance (Z-1)'' versus frequency form 2

Hz to 10 Hz (in nF) plotted:(a) against dielectric constant of the media for a bare and a

silanizednanogap devices, and (b) against -log of [KCl] solutions; for each graph in figure

b, all the slopes are normalized with respect to the slope for -log [KCl]=5. .................... 61

Figure 3. 1 TEM images of 10 mM DDAB solution in DI water (A), 0.5 mM Hb in 10

mM acetate buffer (pH 5.6) (B), and a 1:1 mixture of DDAB with Hb as described in the

experimental section (C); a solution of uranyl acetate was used only for (A) to obtain a

sharp contrast (all scale bars are 5 µm). ............................................................................ 72

Figure 3. 2 Cyclic voltammograms of bare GCE (solid black), DDAB-modified GCE

(dashed gray) and Hb/DDAB-modified GCE (solid red) in 100 mM phosphate buffer

solution (PBS, pH 7.4) with 50 mM NaBr in the absence (A) and presence (B) of 8 mM

sodium nitrite at 100 mV s-1.............................................................................................. 72

Figure 3. 3 Cyclic voltammograms of Hb/DDAB-modified GCEs at various pH in the

absence (A) and presence (B) of 8 mM sodium nitrite in 100 mM PBS (pH 5.8, 6.6, 7.4

and 8.2) buffer with 50 mM NaBr at 100 mV s-1.............................................................. 77

Figure 3. 4 Cathodic (A,C) and anodic (B,D) DPV for Hb/DDAB-modified GCEs in 100

mM PBS (pH 5.8, 6.6, 7.4 and 8.2) buffer with 50 mM NaBr, in the absence (A, B) and

presence of 8 mM sodium nitrite (C, D). .......................................................................... 77

Figure 3. 5 Cathodic (A) and anodic (C) DPV for Hb/DDAB-modified GCE in 100 mM

PBS (pH 7.4) with 50 mM NaBr in the presence of various concentrations of sodium

nitrite; B and D show the magnified images of the merged peaks for redox couples C and

D. ....................................................................................................................................... 79

Figure 3. 6 DPV of Hb-S/DDAB-modified electrode in 100 mM PBS (pH 7.4) with 50

mM NaBr in the presence of various concentrations of sodium nitrite (A), and a plot of

peak current vs concentration of sodium nitrite (B). ........................................................ 85

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Figure 3S. 1 Cyclic voltammograms of Hb-DDAB-modified GCEs in PBS (pH 7.4) with

8 mM nitrite at different scan rates (mV/s) (a). Anodic and cathodic peak currents vs.

square root of scan rate for of Hb-DDAB-modified GCEs in PBS (pH 7.4) with 8 mM

nitrite (b). .......................................................................................................................... 88

Figure 3S. 2 Anodic (an) and cathodic (cat) peak potentials of Hb-DDAB-modified GCEs

vs pH for all peaks. All solutions contained 8 mM nitrite. ............................................... 89

Figure 4.1 Schematic diagram for the electrodeposition of DDAN on a GCE surface (A).

CV of 2 mM DDAN in acetonitrile along with 10 mM of tetrabutylammonium

tetrafluoroborate at a scan rate of 0.05 V s-1 between +0.60 V and -0.90 V vs Ag/AgCl:

scans 1, 2, 29 and 30 (last scan) are shown (B). ............................................................... 99

Figure 4.2 TEM images of a solution of 10 mM of DHP (A, B), and 10 mM of DHP

along with 10 μM of Aβ1-42 (C, D). ................................................................................ 100

Figure 4.3 Schematic illustration of the deposition of the biomimetic membrane on a

GCE surface. The electrodeposition of DDAN on the surface of GCE (A). The

immobilization of DHP was performed by the incubation of DHP vesicles on the surface

overnight (B). .................................................................................................................. 101

Figure 4.4 XPS spectra for the GCE-DDAN-DHP modification showing a clear distinct

phosphorus peak with an asymmetric peak envelop due to the overlapping P2p3/2 and

P2p1/2 spin orbit components. .......................................................................................... 102

Figure 4.5 TOF-SIMS spectra for all three surfaces (500x500 μm2 area): bare glassy

carbon electrode (GCE), the first layer-modified electrode (GCE-DDAN) and bilayer-

modified electrode (GCE-DDAN-DHP) at negative (A) and positive (B) polarity. ...... 103

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Figure 4.6 Cyclic voltammograms and Nyquist plot of the bare GCE, DDAN layer (the

first layer), and DDAN-DHP bilayer. (A) and (B) show the respective CV at a scan rate

of 50 mV s-1 and EIS using [Ru(NH3)6]3+ as the positively-charged redox probe. (C) and

(D) show the respective CV and EIS spectra using [Fe(CN)6]3-/4- as the negatively-

charged redox probe. In Nyquist plots, all dots represent the experimental data and solid

lines represent the simulated data with the applied bias -0.20 V and +0.25 V vs Ag/AgCl

for measurements performed using Ru(NH3)63+ and [Fe(CN)6]

3-/4-, respectively, at a

frequency from 100 mHz to 100 kHz with an amplitude of 5 mV. Insets shows plots for

bare GCE. ........................................................................................................................ 105

Figure 4.7 EIS of modified GCEs in the presence of [Fe(CN)6]3-/4- with EDAN layer (the

first layer) (GCE-EDAN; red circles), and bilayer (GCE-EDAN-BEHP; green circles).

Results are shown before (filled circles) and after (empty circles) stirring with the redox

probe at 500 rpm for 1 min with the applied bias of +0.25 V vs Ag/AgCl at a frequency

ranging from 100 mHz to 100 kHz with an amplitude of 5 mV. .................................... 106

Figure 4.8 EIS of a GCE-DDAN-DHP using [Ru(NH3)6]3+ (A) and [Fe(CN)6]

3-/4- (B) as

the redox probe (green), and after 10 min (purple), 24 h (yellow) and 48 h (blue)

incubation in a solution of Aβ1-42. All dots represent the experimental data and solid lines

represent the simulated data with the applied bias of -0.20 V vs Ag/AgCl with

[Ru(NH3)6]3+ (A) and +0.25 V vs Ag/AgCl with [Fe(CN)6]

3-/4- at a frequency ranging

from 100 mHz to 100 kHz and a potential amplitude of 5 mV. ..................................... 110

Figure 4S.1 Mass spectroscopic characterization of the synthesised diazonium salt

(DDAN, 4-dodecylbenzene-diazonium tetrafluoroborate). ............................................ 113

Figure 4S.2 IR spectra for the synthesised diazonium salt (DDAN, 4-dodecylbenzene

diazonium tetrafluoroborate) and the precursor amine (4-dodecylaniline) (B). ............. 114

Figure 4S.3 Cyclic voltammograms for the electrodeposition of 4-ethylbenzenediazonium

(EDAN) on GCE surface at a scan rate of 50 mV s-1 (refer to Scheme S2). .................. 115

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Figure 4S.4 Cyclic voltammograms and Nyquist plots of bare GCE, GCE-EDAN layer,

and GCE-EDAN-BEHP bilayer. (A) and (B) show the respective CV and EIS spectra

using [Ru(NH3)6]3+ as the redox probe. (C) and (D) show the respective CV and EIS

spectra using [Fe(CN)6]3-/4- as the redox probe. In Nyquist plots, all dots represent the

experimental data and solid lines represent the simulated data using [Ru(NH3)6]3+ and

[Fe(CN)6]3-/4- at an applied bias of -0.20 V and +0.25 V vs Ag/AgCl at a frequencies

range between 100 mHz and 100 kHz with a potential amplitude of 5 mV. .................. 116

Figure 5. 1 Chemical structure of 4-(1-oxa-4,10-dithia-7-aza-cyclododecane) (A) and 4-

(1-oxa-4,10-dithia-7-aza-cyclododec-7-yl) (B). ............................................................. 119

Figure 5. 2 Spectroscopic results for quartz slides: bare and CE-modifed slides (A) and

CE-modifed slide in presence and absence of Hg ions ................................................... 123

Figure 5. 3 CV for four different hemoglobin on DDAB modified GC electrodes ........ 124

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List of Abbreviations

AD Alzheimer’s disease

Aβ Amyloid-β

BEHP Bis(2-ethylhexyl) phosphate

C Capacitance

Cdl Double-layer capacitance

CV Cyclic voltammetry

DDAN 4-dodecylbenzenediazonium

DHP Dihexadecyl phosphate

E or V Potential

EDAN 4-ethyldiazonium

Hb Hemoglobin

Hb-S Hemoglobin Sickle Cell

HBL Electrochemical impedance spectroscopy

ELISA Enzyme-linked immunosorbent assay

GAG Glycosaminoglycan

GCE Glassy carbon electrode

I Current

IR Infrared spectroscopy

L Inductance

MS Mass spectroscopy

NMR Nuclear magnetic resonance

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PBS Phosphate buffered saline

q Electric charge

Rct Charge-transfer resistance

RI Refractive index

Rs Solution resistance

SHBM Slim Hybrid Bilayer Membrane

tBLMs tethered bilayer lipid membranes

THBM Thick Hybrid Bilayer Membrane

TOF-SIMs Time of flight secondary ion mass spectrometry

XPS X-ray photoelectron spectroscopy

Z Impedance

Zw Warburg element

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Chapter 1

1. An introduction to electrochemical sensors and biosensors

1.1 Electrochemical biosensors

A chemical sensor is a device which transforms chemical information into an analytically useful

signal and usually contains two basic components connected in series: a chemical (molecular)

recognition system (receptor) and a physico-chemical transducer.[1–3] A Biosensor is a

chemical sensors in which the recognition system utilises a biochemical mechanism.[3] On the

other hand, an electrochemical sencor/biosensor is a sensor/biosensor with an electrochemical

transducer.[3] In biological and chemical sciences, quantification of molecules taking role in

processes plays an extreme role. However, it is challenging to convert the biological

information to a quantifiable and processable signal. A biosensor is doing this job as a

fascinating analytical device.[4] The first biosensor was built by Clark and Lyons in 1962 to

measure the concentration of glucose.[5]

Figure 1.1 Biocomponent and transducers used in biosensors.[6]

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Biosensors may be classified according to the biological elements used in the construction of

the recognition layer or based on the method of signal transduction. It should be mentioned that

biosensors can be used either for biological or non-biological samples.[3] As depicted in Figure

1.1, biosensors may consist of different recognition layer elements (which control the

specificity and selectivity of a biosensor), and various types of transduction methods.

Recognition elements in biosensors may be divided in three classes: biocatalytic, bio-affinity

and hybrid receptors.[7] There are many different transduction methods like optical, thermal,

piezoelectric and electrochemical transducers; I will discuss the latter one in more detail in the

following paragraphs.

- Amperometric biosensors continuously measure current resulting from the oxidation or

reduction of an electroactive species in a biochemical reaction.[5,8–11] Figure 1.2-A shows

an amperometic response of a biosensor (made by Kueng et al.[10]) to glucose (1 mmol L-1)

followed by four injections of ATP (20 nmol L-1) measured in phosphate buffer at 650 mV in

reference to Ag/AgCl. The change in current is proportional to the ATP concentration as

glucose is consumed at the glucose oxidase (GOD) and hexokinase (HEX) modified

platinum disk electrode.

- Potentiometric biosensors in which Ecell is measured at zero current which is proportional to

the concentration according to the Nernst equation (eq. 1.2.17). Figure 1.2-B shows an

example of a potentiometric biosensor.[12] The detection principle relies on the

hybridization process of single-stranded oligonucleotides close to a PVC membrane, which

induces a measurable redistribution of the ion concentration within intermolecular regions.

- Conductometric biosensors measure changes in conductance of an analyte or a medium as a

response to the concentration change of the target biomolecule. Recently, the interest in

conductometric immunosensors in combination with nanowires has been increased.[13–15]

Figure 1.2-C and 1.2-D shows a schematic representation of a nanowire as a base for a

biosensor made by Janissen et al.[16] and the conductometric response of the biosensor.

- Cyclic and differential pulse voltammetry are also a common analytical techniques for

electrochemical biosensors.[17–20] Figure 1.3-A shows a series of cyclic voltammograms

for a cholesterol oxidase (ChOx) /Prussian blue (PB) sol-gel modified prepared glassy

carbon electrode (GCE) used for detection of cholesterol concentration.[18] Figure 1.3-B

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shows a series of DPV plots along with the corresponding calibration curve for a biosensor

sensitive to miRNA-21.[21]

Figure 1. 2 Ampertometirc response of a biosensor to glucose (1 mmol L-1) followed by four injections of ATP (20

nmol L-1) measured in phosphate buffer at 650 mV in reference to Ag/AgCl (A). Potentiometric detection of the

hybridization with complementary oligo(dT)15 on a PVC membrane functionalized with cholesterol-oligo(dA)15 (B).

A nanowire used as a conductometric biosensor (C) and its response to the titration of complementary target DNA

(red) and noncomplementary probe DNA (blue) (n = 4 each) (D).[10,12,16]

- Impedance spectroscopy is another powerful electrochemical tool for transducing the

response of biosensors.[22–26] Park et al.[27] prepared a biosensor consisting of a gold

electrode coated with erythrocyte membrane to detect fibrinogen. Fig. 1.1.3-C shows an

illustration of such an electrode and Fig. 1.1.3-D shows the impedance spectroscopy results

for this biosensor.

A B

C D

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-

Figure 1. 3 Cyclic voltammograms a cholesterol oxidase (ChOx) /Prussian blue (PB) sol-gel modified GCE (B) in a

phosphate buffer (pH 6.8)_0.1 mol/L KCl_0.8% Triton X-100 for a blank solution(a) and solution with cholesterol 10

(b), 20 (c), 30 (d), 40 (e), 50 (f) nM.at Scan rate 50mV/s (A). DPV curves of an electrochemical biosensor as the

concentration of miRNA-21 varies from 0 (a) to 0.05 (b), 0.1 (c), 0.5 (d), 1 (e), 2 (f), 3 (g), 5 (h), 8 (i), and 10 fM (j).

(B) while the inset is the calibration curve for the miRNA-21 concentration from 0.05 to 5 fM. An illustration of the

gold electrochemical impedance biosensor coated (EIB) with Erythrocyte membrane (EM) and treated with fibrinogen

(Fib) (C) and the related Nyquist plots (D).[18,21,27]

- Field-Effect Transistor (FET)-based biosensors[28], electrochemical techniques in

combination with other techniques like electrochemical surface-plasmon resonance

A B

C D

EIB

EIB-EM EIB-EM-Fib

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(ESPR)[29], electrochemical quartz crystal microbalance (EQCM)[30] and many other

techniques have been employed in connection with electrochemical detection in

biosensors.

-

1.2 An introduction to electrochemical techniques

All materials in this section are inspired by: “Electrochemical Methods : Fundamentals and

Applications by ard, A. J.; Faulkner, L. R.,[31] “Electronics Explained: Fundamentals for

Engineers, Technicians, and Makers” by Frenzel, L. E., [32] and “The Handbook of Graphene

Electrochemistry” by Brownson, D. A. C.; Banks, C. E.[33]

1.2.1 Electrical charge, current and voltage

Atoms are constructed of particles with neutral (neutrons), positive (protons) and negative

(electrons) electric charges. Electric charge (q or Q) is measured in coulombs (C) where the

charge of one mole of electrons is approximately equal to 96485 coulombs (Faraday’s law).

Flow of electrical charged particles (normally electrons; e-) is called electrical current (i or I)

which is measured in amperes (A). One ampere is defined as the unit of electric current in

which one coulomb of electric charge passes the circuit in one second (A≈C/s): 𝑖 = 𝑑𝑞/𝑑𝑡 (eq.

1.2.1)

where, i is the instant current, dq is the electric charge and dt is the time. To create an electrical

current, we need to force electrons to flow using different means of electromotive forces (EMF)

like magnetic field or chemical reactions. As a result, electrons flow from one place (negative

polarity) to another place (positive polarity). Traditionally, the flow of electrical current is

assumed as a flow of positive charges; meaning that it is imaginary flows in the opposite of

direction of electron movement from positive polarity to negative polarity. EMF or Electrical

potential difference is measured in volt (V) and is equal to the work (in joule, J) to be done to

move one electric charge (V≈J/C).

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1.2.2 Electrical elements

There are many different electrical elements. Here, we only review the three passive linear

elements: Resistance, Capacitance and Inductance. For all these elements there is a linear

relationship between voltage and current.

- Resistance

When charge passes a resistor, atomic particles of it resist the current. This resistance is

measured in ohms (Ω) and is defined as volt per ampere (Ohm’s law):

𝑅 = 𝑑𝑉/𝑑𝐼 (eq. 1.2.2)

where, R is resistance of the material or electrical element, dV is the voltage or electrical

potential and dI is the electric current. Figure 1.4 shows the standard symbol for a

resistor.

- Conductance:

A simple ideal capacitor consists of two conductive plates opposing each other with a

nonconductive material in between. When a voltage is applied to a capacitor, electric

charges accumulate on the plates (opposite charges on either of them):

𝐶 = 𝑑𝑞/𝑑𝑉 (eq. 1.2.3)

where, C is the capacitance, dV is the voltage dq is the electric charge stored on either of

the plates. The standard symbol for a capacitor is shown in Figure 1.5

- Inductance:

When a current flow through a conductive material generates a magnetic field around

that material (Ampere’s circuit law). If this current varies, the magnetic field changes.

This change induces a voltage which opposes the applied voltage. Inductance is defined

by: 𝑣 = 𝐿 𝑑𝑖/𝑑𝑡 (eq. 1.2.4)

Figure 1. 4 Standard symbol for a resistor

Figure 1. 5 Standard symbol for a capacitor

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where, v is the instantaneous voltage across the inductor, L is inductance in Henry (H)

and di/dt is the instantaneous current change across the inductor. The standard symbol

for and inductor is shown in Figure 1.6.

Figure 1. 6 Standard symbol for an inductor

1.2.3 Direct current (DC) voltage and alternating current (AC) voltage

- DC Voltage is a voltage which is constant over the time and its magnitude and polarity

does not vary over the time.

- AC voltage is a voltage for which the magnitude and polarity varies as a function of

time. A simple sinusoidal voltage can be mathematically described as below:

𝑉(𝑡) =∣ 𝑉 ∣ sin (ω𝑡) (eq. 1.2.5)

where V(t) is the voltage, ∣V∣ is the voltage magnitude, ω is the angular frequency and t

is time in s. As we know 𝜔 = 2π𝑓 (eq. 1.2.6)

where f is the physical frequency in Hz.

1.2.4 Electrical impedance

Electrical impedance of any electrical component is the opposition of that element to the current

when a voltage is applied. While the resistance is simply the change in the magnitude of the

current, the impedance is change of current in either of the magnitude (for DC and AC voltages)

and phase (for AC voltage). Impedance can be shown by symbol Z. To calculate impedance of

any combination of different electrical elements real numbers can be used, but using complex

numbers makes the calculation much easier. By using complex numbers, we add an imaginary

part to the real values given that at the end of calculation we can remove the imaginary parts

and the results are like when we only use real numbers. Let’s consider the following equation

for a sinusoidal voltage with a phase equal to ϕ:

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𝑉(𝑡) =∣ 𝑉 ∣ cos (𝜔𝑡 + 𝜑) (eq. 1.2.7)

We can use the complex representation for the same voltage as below:

𝑉(𝑡) =∣ 𝑉 ∣ 𝑒𝑗(𝜔𝑡+𝜑) = 𝑉′ + 𝑗𝑉′′ (eq. 1.2.8)

where j is the imaginary unit (j2=-1), V’ is the real part of the voltage and V’’ is the imaginary

part of the voltage.

Impedance (Z) of electrical elements are as below:

For a resistance: 𝑍𝑅 = 𝑅 (eq. 1.2.9)

For a capacitance: 𝑍𝐶 =1

𝑗𝜔𝐶= −𝑗𝜔−1𝐶 (eq. 1.2.10)

For an inductor: 𝑍𝐿 = 𝑗𝜔𝐿 (eq. 1.2.11)

1.2.5 Impedance spectroscopy

By applying an AC voltage (with a known magnitude and phase) over an (or a combination of)

electrical element and measuring the (magnitude and phase) of the resulted current, we can

study the behavior of a real electrical component which could be much complicated compared

to a basic electrical element.

Although, this behavior could be very similar to a combination of the basic electrical elements

which would allow us to simulate/fit that behavior. To understand the behavior of basic

electrical elements, I used a lock-in instrument (Figure 1.7) by which, I could apply a sinusoidal

voltage (Vapp=∣V∣app cos(ωt)). I used different electrical elements (EE) and a known resistor (RG)

to build a voltage divider. A voltage follower was used to measure the voltage (VRG=∣V∣RG

cos(ωt+θ)) across RG.

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Figure 1. 7 Schematic diagram of the circuit used for impedance spectroscopy. Vout is the applied voltage by the lock-

in amplifier, Rint is the internal resistance of the lock-in amplifier, EE stands for any different electrical elements which

is being tested, RG is the gain resistor, and Vin is the voltage across RG.

To calculate the impedance across the EE (ZEE), I used the fact that the current passing the EE is

equal to the current passes RG, and the Ohm’s law. The final equations -are as follows:

𝑍′ = (𝑅𝐺∣𝑉∣𝑎𝑝𝑝

∣𝑉∣𝑅𝐺) cos(𝜃) − 𝑅𝐺 (eq. 1.2.12)

𝑍′′ = − (𝑅𝐺∣𝑉∣𝑎𝑝𝑝

∣𝑉∣𝑅𝐺) sin(𝜃) (eq. 1.2.13)

𝜑 = 𝐴𝑟𝑐𝑡𝑎𝑛(𝑍′′

𝑍′ ) (eq. 1.2.14)

where Z’ and Z’’ are the real and imaginary part of the impedance of EE respectively, ϕ is the

phase of impedance of EE, and θ is the phase of voltage over RG. To depict the impedance

spectroscopy results, there are different ways among which Nyquist and Bode plots are widely

used by chemists. Samples of a Nyquist and a Bode plot for a resistor are shown in Figure 1.8

(C and D), respectively.

Followings are the results for four different combinations for EE:

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Figure 1. 8 A resistor as the “EE” (A). Measured real part (V’, blue) and imaginary part (V’’, red) of voltage over the

RG vs frequency of applied voltage (B). Bode plot of the calculated magnitude (Z) and phase (ϕ) of impedance of “EE”

vs frequency of applied voltage (C). Nyquist plot of calculated imaginary (Z’’) vs real (Z’) part of impedance of “EE”

for the whole range of applied frequency (D).

Figure 1.8-C shows the Bode plot for a resistor 10.03 kΩ. The magnitude and phase of

impedance are almost constant over the applied frequencies and are around 10 kΩ and 0 degree,

respectively. Figure and 1.8-D shows the Nyquist plot for the same circuit and is a point at 10

kΩ on the X-axis meaning that there is no imaginary part for the impedance. It should be

mentioned that there was always a very small deviation from ideality. Some reasons could be

self-capacitance of any components and self-inductance specially at high frequencies.

R = 10.03 kΩ

RG = 10.00 kΩ

EE=

A B

C D

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Figure 1. 9 A capacitor as the “EE” (A). Measured real part (V’, blue) and imaginary part (V’’, red) of voltage over

the RG vs frequency of applied voltage (B). Bode plot of the calculated magnitude (Z) and phase (ϕ) of impedance of

“EE” vs frequency of applied voltage (C). Nyquist plot of calculated imaginary (Z’’) vs real (Z’) part of impedance of

“EE” for the whole range of applied frequency (D).

C = 127 nF

RG = 10.00 kΩ

EE =

A B

C D

Low

frequencies

High

frequencies

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Figure 1.9-C shows the Bode plot for a capacitor 127 nF. While the phase of impedance is

almost constant (around 90 degree) over the applied frequencies, the magnitude of impedance is

huge at low frequencies which diminishes to almost zero at high frequencies.

Figure 1. 10 A resistor and a capacitor in parallel as the “EE” (A). Measured real part (V’, blue) and imaginary part

(V’’, red) of voltage over the RG vs frequency of applied voltage (B). Bode plot of the calculated magnitude (Z) and

phase (ϕ) of impedance of “EE” vs frequency of applied voltage (C). Nyquist plot of calculated imaginary (Z’’) vs real

(Z’) part of impedance of “EE” for the whole range of applied frequency (D).

This can be understood from eq. 1.2.10 where the impedance of capacitors is inversely

proportional to the frequency. Figure 1.10 (C and D) shows the Nyquist plot for the same

C = 127 nF

RG = 1.00 kΩ

EE =

R = 10.00 kΩ

A B

C D

Low

frequencies

High

frequencies

𝜔𝑚𝑎𝑥 = 1/𝑅𝐶

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circuit. All points are almost on the Y-axis meaning that there is no real part for the impedance.

The impedance is only imaginary. The capacitor only changes the phase of current and if it was

possible to have an ideal capacitor with zero resistance, the capacitor would be charged

instantaneously (dt=0) and so the current would be infinite.

A resistor and a capacitor were in parallel for the experiment in Fig. 1.2.7. The Nyquist plot

(Figure 1.10-D) for this circuit is a semicircle starting from 10 kΩ at low frequencies with

almost pure real impedance. That is why the phase in Bode plot (Figure 1.10-C) is almost zero

at low frequencies. This is because at low frequencies the impedance of capacitor is huge and

the current passes almost all through the resistance. Oppositely, at high frequencies the

impedance of the capacitor is so small and almost all the current passes through the capacitor.

Also, because the magnitude of this impedance is small the absolute value of impedance of

whole circuit is almost zero. At medium frequencies (200 Hz in this case), the magnitude of the

impedance of capacitor gets very close to the magnitude of the resistor’s impedance. As a result,

the current splits almost into almost half; one half passes through the resistor producing the real

part of the impedance and the other half passes through the capacitor and produces the

imaginary part of the impedance. This point is close to the peak point of the semicircle in

Nyquist plot. The peak point is the point the two part are exactly equal which happens at a

frequency between 100 Hz and 200 Hz which is not measured here.

The circuit in Figure 1.10-A consist of four elements. At low frequencies the capacitors

dominate in both branches and the impedance is almost only imaginary (phase is 90 degree in

Bode plot Figure 1.11-C and it is huge (Z in Bode plot). This produces a very long and

relatively vertical stem in Nyquist plot in Figure 1.11-D (which is not fully shown for the

semicircle to be seen). Since both resistors are almost the same, as the frequency increases, the

impedance of the bigger capacitor decreases faster and starts passing more current in its branch.

At very high frequencies, the impedance both capacitors get very small and the majority of the

impedance belongs to the resistors. That is why the Nyquist plot ends up to the point close to

the 5 kΩ which is equal to the total resistance of two 10 kΩ resistors in parallel. The semicircle

is a result of both capacitor while the smaller one has a bit more role in the shape of the

semicircle.

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Figure 1. 11 A series of a resistor and a capacitor in parallel with another set as the “EE” (A). Measured real part (V’,

blue) and imaginary part (V’’, red) of voltage over the RG vs frequency of applied voltage (B). Bode plot of the

calculated magnitude (Z) and phase (ϕ) of impedance of “EE” vs frequency of applied voltage (C). Nyquist plot of

calculated imaginary (Z’’) vs real (Z’) part of impedance of “EE” for the frequencies from 100 Hz to 100 kHz; data

for lower frequencies are not shown in order to magnify the semicircle (D).

C2 = 127 nF

RG = 10.05 kΩ

EE =

R2 = 10.15 kΩ

R1 = 10.00 kΩ

C1 = 560 pF

A B

C D

Low

frequencies

High

frequencies

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1.2.6 Electrochemical cell and impedance spectroscopy: constant phase element, Warburg

element, and Randles equivalent circuit

When we use impedance spectroscopy to study electrochemical cells, we may find a circuit

combined of several electrical elements which can (approximately) show the same behavior of

the cell. This circuit is called equivalent circuit of that electrochemical cell. In section 1.2.2, we

reviewed three type of basic linear circuit elements. To simulate and fit impedance plot

resulting from an electrochemical cell, two of those elements (resistance and capacitance) are

widely used. But they are not enough, because there are lots of chemical and physical

phenomena included in an electrochemical cell which makes their impedance behavior

complicated. One simple element which have been used broadly by chemists is constant phase

element (CPE) which can be imagined as a non-ideal capacitor. Several symbols have been used

to show CPE in literature; one of them is shown in Figure 1.22-A.

The impedance of a CPE can be represented by the following formula:

𝑍𝐶𝑃𝐸 =1

(𝑗𝜔)𝛼𝐶= (𝑗𝜔)−𝛼𝐶𝐶𝑃𝐸 (eq. 1.2.27)

Comparing this equation with eq. 1.2.10 shows that for α=1 CPE will be an ideal capacitor.

A very common equivalent circuit for electrochemical cells which is abundantly in use by

chemists is Randels equivalent circuit (Figure 1.22-B). The current is a combination of two

contributors: faradaic and non-faradaic current which were discussed in section 1.2.7. Rs and Cd

are previously in section 1.2.8 as solution resistance and double layer capacitance. Rct which is

the charge transfer resistance (section 1.2.8) and a new element Zw which is called Warburg

element are in series and pass the faradic part of the current. Zw represents a kind of resistance

to mass transfer and can be formulated as: 𝑍𝑤 = 𝜎𝜔−1/2 − 𝑗𝜎𝜔−1/2 (eq. 1.2.28)

where σ is a constant parameter depending on several variations including the diffusion

coefficient of oxidant and reductant species on the electrode.

As we understand from eq. 1.2.28, the Warburg element consists of two (real an imaginary)

parts which are equal (in ideal case) and both decrease as the frequency increase. That is how

the low frequency part of the impedance (mass transfer region in Figure 1.22) appears as a line

with 450slope in high values region. The semicircle part can be imagined as a combination of

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16

Figure 1.9 and 1.10 which is a result of sum of responses of solution resistance (RS), charge

transfer resistance (Rct) and double layer capacitance (Cd) to the change of frequency

Figure 1. 12 A symbol for constant phase element (A). Randles equivalent circuit (B). A typical

Nyquist plot for an electrochemical cell.[31]

A

C

B

Rs

Rs Rs Rs

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Figure 1. 13 Schematic diagram of a typical electrochemical cell. The power supply applies a voltage difference

between the two electrodes. The voltmeter and the ammeter measure the voltage (potential, E) between the electrodes

and the current (i) passes the cell, respectively (A). the current-potential (i-E) curve for the electrochemical cell.[31]

A

B

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1.2.7 Electrochemical Cell

Figure 1.12-A shows a typical electrochemical cell which can be depicted as follows:

𝑃𝑡 ∣ 𝐻+, 𝐵𝑟− (1𝑀) ∣ 𝐴𝑔𝐵𝑟(𝑠) ∣ 𝐴𝑔

The possible oxidation and reduction reactions on the Ag electrode are as follows:

𝐴𝑔𝐵𝑟 + 𝑒− 𝐴𝑔 + 𝐵𝑟− 𝐸0 = 0.0713 𝑉 𝑣𝑠 𝑁𝐻𝐸

This chemical equilibrium shows that the Ag electrode is at an equilibrium potential. On the

other hand, for the Pt electrode, there is no ox/red couple present. We may consider the two

possible oxidation and reduction reactions as below:

2𝐻+ + 2𝑒− → 𝐻2 𝐸0 = 0 𝑉 𝑣𝑠 𝑁𝐻𝐸

2𝐵𝑟− → 𝐵𝑟2 + 2𝑒− 𝐸0 = +1.09 𝑉 𝑣𝑠 𝑁𝐻𝐸

This means that: although the open-circuit potential (OCP) does not exist here, we can consider

it between the two potentials near the E0 for the above reactions. While the potential is between

these two values, no current exists. If we apply a voltage in a way that the potential on Pt goes

toward values less than -0.07V vs Ag/AgBr, a reduction reaction (of H+) happens and a cathodic

current passes the Pt electrode. If the potential goes toward values greater than +1.02V

Ag/AgBr, oxidation of Br- occurs on Pt electrode and thus, an anodic current flows through the

Pt electrode. All of these depicted in Figure 1.12-B.

There are two types of electrochemical cells:

- Galvanic cell: a cell in which the reaction on both electrodes are spontaneous when the

electrodes are connected externally by a conductive material.

- Electrolytic cell: a cell in which the reactions at cathode and anode are affected, if an

external source of voltage applies a potential difference (greater than the open circuit

potential) between the two electrodes.

1.2.8 Faradaic and non-Faradaic processes

When a reaction occurs at the electrode surface and electrons pass the electrode-solution

interface (charge transfer), the process is called Faradaic process (follows Faraday’s law) and

the current is called Faradaic current. In cases that no charge passes the electrode-solution

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interface like adsorption, desorption and double layer charging as a result of changes in

potential or current, the process is called non-Faradaic. The resulted current in these cases may

be transient. These two types of processes may happen at the same time.

Figure 1. 14 A typical model of electrical double layer.[31]

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Figure 1. 15 Two-electrode cell (A) with its equivalent circuit (B). A potential step (C) which results in a transient

current i (D).[31]

1.2.9 Electrical double layer capacitance and voltage step

When an electrode is charged, the ionic structure of the solution around the electrode changes. T

his change is a response to the electric field because of the electric charges (positive or

negative) accumulated on the electrode surface. The ions with opposite charges migrate toward

the electrode and the ones with same charge are repelled outward. As a result, a charge

separation occurs in the solution close to the electrode surface which can be imagined like a

capacitor. The region that the charge separation happens is called electrical double layer. Figure

1.13 depicts a typical electrical double layer in which some anions (could be cations) and

solvent molecules are specifically adsorbed on the electrode surface. The locus of the centers of

A

B

C

D

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these ions are considered as inner Helmholtz plane (IHP). The solvated cations are attracted to

the negatively charged surface and construct the diffuse layer. The locus of the centers of the

closest ions with opposite charge to the electrode surface is considered as outer Helmholtz plane

(OHP). The sum of the charge density of specifically adsorbed ions in IHP (σi) and the one of

the ions in the three-dimensional diffuse layer (σd) is exactly equal to the charge density on the

electrode surface (σM).

To study the charging current of a diffuse layer, we may imagine a cell (Figure 1.14-A)

consisting an ideal reversible electrode (like SCE) and an ideal polarized electrode (IPE) at

which for a given potential range there is no faradaic charge transfer on the electrode and there

is only non-Faradaic current for the diffuse layer capacitance charging. This system can be

represented as a double layer capacitance (Cd) of the IPE in series with a resistance (RS)

resembling the all solutions and membrane resistance in the cell as well as the double layer

capacitance on the SCE (CSCE). Since the CSCE is much larger than the Cd, we may neglect the

CSCE and consider a simple RC circuit (Figure 1.14-B) as an equivalent circuit of the cell.

If we apply a voltage step (E; Figure 1.14-C) to this cell and measure the current (Figure 1.14-

D), the resulted transient current (i) as time (t) passes can be mathematically shown as:

𝑖 =𝐸

𝑅𝑠(𝑒

−𝑡

𝑅𝑠𝐶𝑑) (eq. 1.2.15)

1.2.9 Factors affecting electrode reaction rate and Nernst equation

Let’s consider a general half electrochemical reaction which occurs on an electrode in the cell

as:

𝑂 + 𝑛𝑒− 𝑅 (eq. 1.2.16)

where O, R and n are the oxidant, reductant and the number of transferred electrons

respectively. The resulted current of this electrode is governed by several phenomena (refer to

Fig. 1.2.12-A):

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Figure 1. 16 Pathway of redox reactions on electrode (A) and the equivalent circuit (B).[31]

Rct Rrxn Rmt

A

B

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- Mass transfer (of oxidant and reductant from the bulk solution to the electrode surface

and vice versa

- Electron transfer at the electrode surface

- Different chemical and surface reactions following or proceeding the electron transfer

Each of the above phenomena can be described by a resistance element as depicted in Figure

1.15-B, where Rct, Rrxn and Rmt represent the charge transfer resistance, the chemical reactions

resistance and mass transfer resistance, respectively. For a general electrode reaction like in eq.

1.2.16, we can rely on Nernst equation:

𝐸 = 𝐸0 +𝑅𝑇

𝑛𝐹ln (

[𝑂]

[𝑅]) (eq. 1.2.17)

where E and E0 are the actual and standard electrode potentials, R is the universal gas constant,

T is the absolute temperature of the cell, n is the number of electrons in charge transfer reaction,

[O] and [R] are the oxidant and reductant activity.

1.2.10 Controlled potential experiments: Linear sweep voltammetry, cyclic voltammetry and

differential pulse voltammetry

To study electrochemical reactions, we normally focus on just one electrode (working electrode,

WE) in the cell and have an auxiliary electrode (AE) with a large area which provides a

complementary half reaction for the WE while has the smallest possible

Figure 1. 17 A general set-up for three-electrode cell to run controlled-potential measurements.[31]

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Figure 1. 18 A potential step applied to an electrode (A), the resulted current (B) and the concentration profile for

various times (C).[31]

A

B

C

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charging current and other effects on the cell behavior. The potential is applied between these

two electrodes and the current is measured between these two. To control the potential on the

WE with respect to a known reference potential, electrochemists use a reference electrode (RE)

to construct a three-electrode cell (Figure 1.16). One example for RE is Ag/AgCl electrode.

We may assume that a reaction like in eq. 1.2.16 occurs on the WE in Fig. 1.16 and we apply a

potential step between WE and AE (Figure 1.17-A) to satisfy a desired potential step between

WE and RE and measure the current (i). The electroactive specie in this case is an oxidant (O)

which is not electroactive at E1 but is reduced at a diffusion-limited rate at E2. The response to

this potential change, is current. A typical current would be similar to the Figure 1.17-B which

is a combination of capacitance charging current (section 1.2.8) and a charge transfer current.

Chemists are normally interested in the later one. This current is a function of time because of

changing the concentration of electroactive species as the reaction proceeds (Figure 1.17-C). So,

to get the best signal, we need to pick up a point on the curve (Figure 1.17-B) at which the

charging currents (non-Faradaic) is small enough and the charge transfer (Faradaic) current is

large as possible. the diffusion-controlled current (point 4 and 5 in Figure 1.17-C) and E1/2 are a

characteristic of the electroactive species in the given chemical and physical condition of the

cell. If we apply a series of potential steps (Figure 1.18-A) and sample the current at a certain

time after applying each potential step (Figure 1.18-B) and plot the sampled currents (iτ) versus

the potential (E), the resulted curve is called sampled-current voltammogram (Figure 1.18-C). It

should be mentioned that it is assumed that between the potential steps the solution gets

homogenized and the amount of the electroactive specie consumed because of the reaction is

negligible compared to the bulk concentration and so the initial concentration on the electrode

surface can be considered constant at the start of each step.

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Figure 1. 19 A series of potential steps applied to a working electrode (A), the current-time curves (B), the sampled-

current voltammogram(C), a potential sweep (D), i-t-E surface for a Nernstian reaction (E), linear sweep voltammetry

across this surface (F).[31]

If we apply a potential sweep (Figure 1.18-D) instead of a series of potential steps without

renewing the surface concentration and plot the current versus the potential for all times, the

Id

A

B

C

D

E1/2

E

F

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result would be i-t-E surface as Figure 1.18-E. For a given sweep rate, the i-E curve would be a

curve like the intersection of a plane crossed the i-t-E surface which is called a linear sweep

voltammetry or LSV (Figure 1.18-F). If we sweep the potential in one way and at a (switching)

potential (Eλ) reverse the sweep (Figure 1.19-A), the resulted current- potential curve is called

Cyclic voltammetry (CV) (Figure 1.19-B).

Figure 1. 20 Cyclic potential sweep (A), resulted cyclic voltammogram (B), pulse superimposed staircase potential

(C), and the resulted differential pulse voltammogram.[31,33]

FWHM

Ep/2

A B

C D

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Instead of a sweep potential in linear sweep voltammogram, we may apply a staircase potential

with a small pulse at the end of each step (Figure 1.19-C) and measure the current right before

the pulse (I1) and right before the end of pulse (I2). If we plot the difference between each I1 and

I2 couple (ΔI) versus the potential, the result will be a differential pulse voltammetry or DPV

(Figure 1.19-D) which is a physical differential of the related linear sweep voltammetry.

Let’s consider a typical reaction like in eq. 1.2.16 and define the reaction rate constant for the

forward and backward reactions as kf and kb:

𝑂 + 𝑛𝑒− 𝑅

Here, we assume that this reaction is a one-step reversible Nernstian reaction. It has been shown

that if the switching potential (Eλ in Figure 1.19-C) is far enough from the peak potentials (more

than 200 mV) for peak-to-peak separation (Eox and Ered respectively) we have:

𝛥𝐸𝑝 = 𝐸𝑃𝑜𝑥 − 𝐸𝑃

𝑟𝑒𝑑 ≈ 2.218𝑅𝑇/𝑛𝐹 (eq. 1.2.18)

This equation works for reversible reaction. In case of irreversible (or semi-reversible)

reactions, the 𝛥𝐸𝑝 value depends on the scan rate (ν). The larger the scan rate the larger the

𝛥𝐸𝑝. In DPV we can define full-width at half-the-peak-maximum height (FWHM; Figure 1.19-

D). It has been shown that: 𝐹𝑊𝐻𝑀 ≈ 3.53𝑅𝑇/𝑛𝐹 (eq. 1.2.19)

Also, it is shown that for EP/2 (Figure 1.19-C) which corresponds to the potential at which half

the peak current is observed we have:

𝐸𝑝 − 𝐸𝑝/2 ≈ 2.218𝑅𝑇/𝑛𝐹 (eq. 1.2.20)

This equation can be used for both ox and red peaks. It is worth to remember that the symmetry

in CV peak depends on several parameters. One of them is the scan rate which was mentioned

above. Another one is called transfer coefficient (α) which shows up in the calculation of kf and

kb as follows: 𝑘𝑓 = 𝑘0𝑒(−𝛼𝐹

𝑅𝑇(𝐸−𝐸0))

(eq. 1.2.21)

𝑘𝑏 = 𝑘0𝑒((1−𝛼)𝐹

𝑅𝑇(𝐸−𝐸0))

(eq. 1.2.22)

kf

kb

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Figure 1. 21 Theoretical cyclic voltammogram showing the effect of ψ and α. Curve 1: ψ=0.5 and α=0.7, Curve 2:

ψ=0.5 and α=0.3 (A). Curve 3: ψ=0.7 and α=0.5, Curve 1: ψ=0.25 and α=0.5. (Note E1/2=EP/2).[31]

Where k0 is the standard rate constant. The larger the rate constant the faster the reaction. As we

can understand from these two equations, the value of transfer coefficient (α) which is between

0 and 1, determines the ratio of 𝐼𝑃𝑜𝑥 and 𝐼𝑃

𝑟𝑒𝑑 (Figure 1.20-C). In the case of α=0.5, there is

symmetry and the ratio (𝐼𝑃𝑜𝑥/𝐼𝑃

𝑟𝑒𝑑) is 1. The standard rate constant (k0), also

appears in Butler-Volmer formulation of electrode kinetics as below:

𝑖 = 𝐹𝐴𝑘0[𝐶𝑜𝑥(0, 𝑡)𝑒(−𝛼𝐹

𝑅𝑇(𝐸−𝐸0)) − 𝐶𝑟𝑒𝑑(0, 𝑡)𝑒(

(1−𝛼)𝐹

𝑅𝑇(𝐸−𝐸0))

(eq. 1.2.23)

This formulation shows that for a larger rate constant, the peak current will be larger. To gather

the effect of all affecting parameters which define the shape of a cyclic voltammogram, one can

use a dimensionless parameter (ψ) as below:

𝜓 =(𝐷𝑜𝑥/𝐷𝑟𝑒𝑑)

(𝛼2

)𝑘0

(𝜋𝐷𝑜𝑥𝐹𝜈

𝑅𝑇)

12

(eq. 1.2.24)

A B

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where 𝐷𝑜𝑥 and 𝐷𝑟𝑒𝑑are the diffusion coefficient of ox and res species respectively. Figure 1.20

shows the effect of ψ (which represents all affecting parameters: Dox, Dred, α, k0, ν) on a cyclic

voltammogram.

1.2.11 Multicomponent systems and multi-step charge transfer

Consider two consecutive peaks in a cyclic voltammetry experiment related to two one-electron

electrochemical reactions. Figure 1.21 shows some calculated CVs for different ΔEP (EP,1-EP,2).

In part A of this figure, ΔEP=-180 mV which makes the two peaks distinguishable, although it is

not easy to define a baseline for the second peak. For cases with ΔEP between 0 and -100 mV

(Figure 1.21-B) two peaks are merged into a broaden peak and the EP does not change by

varying the scan rate.

Figure 1. 22 Calculated CVs for two reversible one-electron steps system where n2=n1=1 at 25oC. ΔEP is equal to -

180mV, -90 mV, 0 mV and +180 mV ofr (A). (B), (C) and (D), respectively.[31]

A

B

C

D

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When ΔEP=0 mV (Figure 1.21-C) two peaks completely merged and shows a behavior between

the characteristics of 1-electron and two-electron reactions. If ΔEP is positive and large enough

(≥+180 mV) like the one in Figure 1.21-D the two peaks are behaving like a one-step two-

electron reaction.

1.2.12 Effect of pH on peak potentials

Let’s consider a typical fully electrochemically reversible reaction consisting a certain

concentration of hydronium ions:

𝑂 + 𝑚[𝐻+] + 𝑛𝑒− 𝑅 (eq. 1.2.25)

The relevant Nernst equation would be as follows:

𝐸 = 𝐸0 +𝑅𝑇

𝑛𝐹ln (

[𝑂].[𝐻+]𝑚

[𝑅]) = 𝐸0 + 2.303

𝑅𝑇

𝑛𝐹log (

[𝑂]

[𝑅]) − 2.303𝑚

𝑅𝑇

𝑛𝐹𝑝𝐻 (eq. 1.2.26)

This equation shows that peak potential of the example reaction changes as the pH changes by a

slope of -2.303mRT/nF.

1.2.13 Calculation of number of electrons for an electrochemical reaction using CV and DPV

In section 1.2.10, we reviewed three different equations from which we can calculate the

number of electrons (n) for an electrochemical reaction. Table 1.1 summarizes these equations.

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Table 1.1 Methods used for calculation of number of electrons for known electrochemical reactions

Method Electrochemical

approach

Equation used

1 CV ΔEp = EPox − EP

red ≈ 2.218RT/nF (eq.

1.2.18)

2 CV Ep − Ep/2 ≈ 2.218RT/nF (eq.

1.2.20)

3 DPV FWHM ≈ 3.53RT/nF (eq.

1.2.19)

I tried to find the best method in our laboratory for the two well-known electrochemical

material K3Fe(CN)6 and Ru(NH3)6Cl3, both dealing with one electron (Fe(CN)63-/ Fe(CN)6

4- and

Ru(NH3)62+/ Ru(NH3)6

3+). For all experiments we used a gold screen printed electrode in an

aqueous solution of 5 mM of the related material consisting 50 mM of NaBr with Autolab

potentiostat/galvanostat model 737 Metrohm (Utrecht, the Netherlands). All solutions were

purged for at least 3 minutes by nitrogen before each experiment. All salts were purchased from

Sigma-Aldrich (Oakville, ON) and used upon receipt. Deionized water was made by a MilliQ

instrument in the lab and was filtered before usage.

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Table 1.2 Calculated number of electrons for known electrochemical reaction using methods in

Table 1.1

Material Scan rate

for CV

Method 1 Method 2

for

anodic

peak

Method 2

for

cathodic

peak

Method 3

K3Fe(CN)6 100 mV/s 0.58 0.93 0.83 0.81

30 mV/s 0.71 1.01 0.99

Ru(NH3)6Cl3 100 mV/s 0.61 0.9 0.92 0.81

30 mV/s 0.75 1.00 1.04

My data (Table 1.2) showed that method 2 which uses the 𝐸𝑝 − 𝐸𝑝/2 values of CVs worked best

at reletaively low scan rates (Here 30 mV/S). So, I decided to use this method for further

investigations in my projects.

.

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1.3 An introduction to Bilayer and Surfactant membranes and chemistry of diazonium

salts used as electrode surface modification in electrochemical sensors

1.3.1 Bilayer membranes

Biological membranes perform a critical role in all living organisms by making

compartmentalization of the biomolecules in cells ensuring homeostasis, material uptake,

energy regulation, signaling and molecular recognition. [34–36] Figure 1.23 illustrates a model

of biological membrane as originally proposed by Singer and Nicolson in 1972.[36]

Figure 1. 23 The fluid mosaic membrane model of a biological membrane structure.[36]

Transmembrane proteins implanted into the bilayer membranes or transiently associated with it,

are key factors in a plethora of cellular processes including intracellular signaling, anchoring of

cytoskeletal proteins or transporting ions and nutrients across the membrane. [37]

Direct investigation of biological membranes is really challenging for scientist, because of the

complexity of biological membranes with different possible components they might have

imbedded. That is why scientists try create artificial bilayers to mimic the cell membrane to

simulate the basic functions of a cell membrane.[38] Solid-supported lipid membranes

(SLBs)[39,40], hybrid bilayer lipid membrane,[41,42] suspended lipid bilayer,[43–45] and

tethered bilayer lipid membranes (tBLMs),[38,46–48] are some examples of these efforts. An

example of tethered bilayer lipid membranes on a gold electrode is illustrated in Figure 1.24.

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Figure 1. 24 A gold electrode with a β-mercaptoethanol SAMs (A) and the formed tethered bilayer lipid membranes

using cholesterol/DOPC (phospholipids,1,2-dioleoyl-sn-glycero-3-phosphocholine) as the second layer (B).[38]

This tBLMs is prepared on gold coated glass slide immersed in a solution of β-mercaptoethanol

to form SAMs (Figure 1.24-A). After incubation, the slides are washed in ethanol and dried in a

stream of nitrogen gas and tBLMs are formed by incubating the SAMs with a solution of

cholesterol/DOPC (phospholipids,1,2-dioleoyl-sn-glycero-3- phosphocholine) (Figure 1.24-B).

The formed tBLMs were verified by electrochemical impedance spectroscopy (EIS) to evaluate

their stability and physical properties.[38]

1.3.2 Surfactant membrane and films

Surfactants have been used for synthetic double layer membranes. These membranes exhibit

interesting properties due to their complex lipid composition. On the other hand, study of

A

B

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proteins as a very important class of biomolecules has been always in interest. Proteins can go

into and out of the organic cells through the cell membrane. That is why synthetic membranes

has been employed to investigate membrane -protein interactions. Surfactant membranes-

proteins (SP) interactions can be classified into two categories: SP-A and SP-D (hydrophilic)

and SP-B and SP-C (hydrophobic), three of which are illustrated in Figure 1.25.[49–52]

Figure 1. 25 Schematic representation (not to scale) of the structure of surfactant proteins SP-A, SP-B and SP-C, and

their mode of interaction with surfactant monolayers and bilayers.[52]

Bilayer membranes have been studied using many different tools. One of the analytical tools we

are using in our lab is electrochemical ones. So, we are interested in membrane and films which

are ready to be prepared on electrodes surface. Some of surfactnts that have been used to

prepare films are shown in Figure 1.26. In 1994, Rusling and Howe50 reported how they used

Didodecyldimethylamonium bromide DDAB) film on poly graphite electrodes for

electrochemical investigations and proposed electron tranfer mechansim through the film.

Rusling and others[53–55] reported an intersting electrochemical tools for investigation of

Myoglobin using DDAB film depostied on electrodes. This approach has been a useful tolls for

studying other proteins by scientists. DDAB film has been also used for studying other

biomacromolecules.[56–58]

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Figure 1. 26 Schematic representation (not to scale) of the structure of surfactant proteins SP-A, SP-B and SP-C, and

their mode of interaction with surfactant monolayers and bilayers.[59]

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1.3.3 Diazonium salt chemistry

Chemistry of diazonium salts has been a subject of continued interesting areas and extensively

developed for years.[60,61] One of the interesting application of this category of reactive

chemicals is modifying surfaces by a wide variety of molecular structres.[62] Save´ant and

Pinson[63] were the first to demonstrate that the reduction of an aryl diazonium cation produces

the corresponding aryl radical, which can further react with a carbon atom of the carbon substrate

to harvest the covalent bonding of this aryl group (Figure 1.27-A).

Among all possible types of chemical reactions, we are also interested in electrochemical ones in

our lab. Electrochemical reduction of diazonium salts (either by using pre-synthesized salts or in

situ[64] syntheses) is a convenient approach for modifying conductive surface like platinum[65],

gold[66,67], iron[68] and specially carbon[63,69–75] electrodes. A typical schematic of

reduction of diazonium salt on conductive surfaces is shown in Figure 1.17-A. Figure 1.17-B

shows CVs for an electrochemical reduction of a diazonium salt on a glassy carbon electrode.[76]

It should be mentioned that electrochemical reduction of diazonium salts on the electrode surface

does not necessarily result in a monolayer of molecules on the electrode. It is quite possible to

have a multilayer on the electrode surface.[77,78] Figure 1.28 shows a schematic illustration of

the predicted mode of such a multilayer. Modified electrodes using diazonium salts have been

recently employed for a wide variety of applications like: study of peptides[79], study of

enzymes[80,81], heavy metal detection[82], neural investigation[83], supercapacitors

developement[84] and hormone immunosensors[85]. Electrografting of aryl layers can be

achieved by dissoling a diazonium salt in an aprotic medium with a supporting electrolyte or in

an acidic aqueous medium and then reducing it at room temperature on the cathode (the surface

to be modified). In addition to this method, diazonium compounds can be produced in situ from

the parent anilines. Figure 1.28 illustrates the complex surface modification of materials by

diazonium compounds resulting in an oligomerized structure with possible azo bonds within the

layer or at the substrate–aryl layer interface.[86] Baranton and Be´langer showed that the layer

obtained in aqueous medium could be slightly thinner or less homogeneous than the one obtained

in acetonitrile.[64]

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Figure 1. 27 A typical schematic for electrochemical reduction of diazonium salt on conductive surfaces (A). A Glassy

carbon disk electrode (d 5 3 mm) in ACN + 2 mM +N2C6H4NO2 BF4- + 0.1 M NBu4BF4, (a) first and (b) second scan

and (c) the same electrode in ACN + 0.1 M NBu4BF4 (B). [62]

Figure 1.29 shows that high grafting density of aryl groups leads to the formation of a stretched

brush layer of controlled structure, with high grafting density, lateral homogeneity, and

activatable end-chains. Aryl diazonium compounds, given their versatility in forming reactive

and functional platforms facilitates the exploration of new chemistry options for surface

treatments, particularly grafting macromolecules and metallic nanoparticles in very mild

conditions.[86]

A

B

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Figure 1. 28 An illustration of the multilayer formation during electrochemical grafting of aryl radicals in solution to the aryl moieties bound to the electrode surface. C = carbon; SC = semi-conductor; M = metal; P = polymer; I = insulating.[86]

A

B

Figure 1. 29 AFM image of PNIPAM-coated gold substrate after 120 min of polymerization and after careful removal of the brush from the Au/Cr layer using a razor blade. The inset compares the XPS survey scans of the gold substrate before and after PNIPAM coating: one can note a complete screening of the Au4f doublet from gold concomitantly with the appearance of the C1s, N1s and O1s peaks from PNIPAM graft. (A) and High resolution TEM image of a MWCNT-grafted PMMA using aryl surface initiators (adapted from (B).[86,87]

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1.4 Overview of next chapters

In all my PhD projects, I concentrated on applying electrochemical techniques in combination

with electrodes which I modified their surfaces using different techniques and employed them

mostly for studying proteins. In chapter 2, I explained how I used impedance spectroscopy to

reveal the electrochemical response of a nano well/nanogap electrode. As a result, I proposed

how this electrode can be used as a capacitive electrode to study different target molecules/ions

by modifying its surface with appropriate recognition element(s). I showed how we can isolate

the capacitive response of the nanogaps and proposed an equivalent circuit which can simulate

the impedimetric behaviour of the electrode in a wide range of frequency (1 Hz to 100 kHz) in

different media with a very wide range of dielectric constant (1 to 110). In chapter 3, I used a

didodecyldimethyl ammonium bromide (DDAB) liquid crystal film to immobilize hemoglobin as

a well-studied protein to investigate nitrite reductase activity of this protein. I employed different

electrochemical techniques to propose -for the first time as of the best of our knowledge- a

complete mechanism of the electrochemical reaction happens on the surface. I showed how this

modifying technique in combination with electrochemical techniques can be used for comparing

the nitrite reductase activity of different type of hemoglobins. My results reveled that the

outcome of this relatively fast techniques is in agreement with well stablished spectroscopic

techniques. I introduced a novel method for modifying glassy carbon electrodes using diazonium

salts and prepare a bilayer as a platform for studying proteins in chapter 4. This method is quite

versatile to design a bilayer with characterizations which may be more suitable for the

protein/biomolecule of interest by choosing appropriate molecular structure for the first and the

second layer used for modifying the electrode surface. This platform an be used for both

electrochemically active and inactive (by using electrochemical probes) targets. Finally, in

chapter 5, I tried to stablish a roadmap for continuing these works. I illustrated how I synthesised

a recognition element which can be used for modification of the nanogap electrode for mercury

ions as a target. I, also, showed how the DDAB modified GCE in combination with DPV can be

used for nitrite reductase activity of different natural and synthetic hemoglobins. In addition, I

proposed some molecular structures as candidates for preparing bilayers with different chemical

affinity for interaction with selected biomelcues.

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Chapter 2

2. Microfabricated, silicon devices with nanowells and nanogap electrodes: a

platform for dielectric spectroscopy with silane-tunable response

2.1 Connecting text

Nanostructures-based devices has been employed to develop chemical sensors with improved

sensitivity, selectivity and possibilities for integration with other tools. [88–90] Nanotubes,

nanorods, nanobelts, nanowires and nanogaps are some these nanostructure that has been used for

this puprposes.[90,91] Electrochemical techniques provide an attractive means to analyze the

content of a chemical and biological samples using nanosensors, due to the direct conversion of a

chemical/biological events to an electronic signal. Any architecture of nanostructures provides

with its own advantages which makes them interesting for developing sensors based on those

structures. In this chapter we describe how we used electrochemical tools to reveal how we could

isolate the response of a novel nanogap structure by means of electrochemical impedance

spectroscopy. Our results show that this nanogap device can be used as a capacitive sensor for

analytes which can change the dielectric constant in a nanogap (gap size: around 40 nm gap).

This work has been published in the following article in collaboration with Yoshinori Suganuma

which developed the nanogap device in Professor Al-Amin Dhirani’s lab. I thank them for

providing me with their vast knowledge of nanostructures, electronics and physics as well as all

tools and materials needed for this project.

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- Fini, H. S.; Suganuma, Y.; Dhirani, A. A. Microfabricated, Silicon Devices with

Nanowells and Nanogap Electrodes: A Platform for Dielectric Spectroscopy with Silane-

Tunable Response. Mater. Res. Express 2015. https://doi.org/10.1088/2053-

1591/2/5/055012.

Author contributions:

Yoshinori Suganuma and Al-Amin Dhirani developed the nanogap device, Hamid Fini and

Yoshinori Suganuma developed the lab made electronic structures and Hamid Fini performed all

the experiment. Hamid Fini and Al-Amin Dhirani discussed the results and wrote the paper.

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2.2 Abstract

Combining the advantages of nanogap devices and impedance spectroscopy can potentially provide

a platform for dielectric spectroscopy with widely ranging applications - from fundamental studies

at the nanoscale and surfaces to label free and selective sensors. The present study characterizes

the impedance response of a microfabricated, silicon-based device with a large array of nanowells

surrounded by annular, nanogap detection regions. Device impedance is measured vs. frequency

over 5 orders in a variety of organic solvents with dielectric constants ranging over 2 orders. The

study finds two key results. First, an equivalent R/C circuit model is found to compare favorably

with device impedance response over these wide ranges of parameters. Importantly, the model

correlates with structure of the nanogap device, which suggests that such a structure-impedance

response approach can help guide modeling of other devices geometries. Second, the model points

to - and data confirm –correlation between nanogap device response and dielectric constant of

materials in the nanogaps, particularly at low frequencies. In addition, the correlation is

significantly modified by robust, silane functionalization of the devices due a large surface-to-

volume ratio of the nanogaps. These results demonstrate that nanogap impedance spectroscopy

using microfabricated/silanized silicon devices is a robust and versatile platform for dielectric

spectroscopy of materials on the nanoscale and on surfaces.

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2.3 Introduction

Nanogap electrodes[92] represent a target of opportunity for next generation sensors and

detectors.[93–98] A feature of nanogaps is that they provide a very sensitive platform; as a result,

they require remarkably small quantities of samples, which is especially desirable for bio-

applications.[99,100] In addition, nano-architectures can possess large surface-to-volume

ratios[101] that in turn generate a high surface sensitivity.[102] Since surfaces of nanogap

electrodes can be modified via functionalization, they provide a means to detect target species

selectively.[97,103–107] Nanogap electrodes can be designed to operate inside the electrical

double layer, and therefore, the background capacitance arising in ionic solutions is substantially

reduced. A consequence is that nanogap electrodes can be more sensitive to capacitive changes

arising from sample permittivity/dielectric constant.[108] Nanogap electrodes have been

successfully used to detect a wide range of chemical species such as DNA[95,108] , proteins[109]

and heavy metals[110].

Many different approaches have been used to convert interactions between nanogap

electrodes and target species into signals. Impedance spectroscopy is one of the most widely used

since it is sensitive, straightforward and cost effective.[88,110–114] The basis of this method is

that a sinusoidal voltage (V) is applied across a linear device, and the resulting current (I) is given

by

I=V/Z (equation 2.1)

where Z is the device’s impedance. The mathematical analysis of the device’s response is

simplified by introducing complex components to the voltage and current, and the impedance is

given by:

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Z=|Z|exp(iφ)=Z’+iZ”, (equation 2.2)

where i=(-1)1/2, Z’ and Z” are real and imaginary parts of the impedance, respectively,

|Z|=(Z’2+Z”2)1/2 is the amplitude, and φ=tan-1(Z”/Z’) is the phase. Z can be calculated in many

instances of practical interest.[115–117]

Models for nanogap electrodes can play an important role in helping guide the design and

application of such devices. Models can guide device design via potential structure-device response

relationships, optimization of device signal e.g. via choice of detection frequency, interpretation of

response e.g. conductivity vs. dielectric contributions, etc.[118–121] The present study develops

and tests an equivalent R/C model for a nanogap device guided by the device’s architecture. The

tests employ a microfabricated, silicon-based architecture with nanowells surrounded by nanogap

annular electrodes designed to generate enhanced sensitivity to the impedance changes by means

of a correspondingly large area (order of mm2) and a small gap size (order of nm).[122,123] The

tests also employ straightforward electronics with a voltage divider configuration which is shown

to possess a wide bandwidth and to simplify analysis of the impedance response of the nanogap

device. The model indicates and data in the present study confirm that, by operating at low

frequencies where the impedance varies linearly with nanogap capacitance, the device is sensitive

to dielectric constant of material in the nanogap regions. All materials possess a dielectric constant,

and in this sense dielectric constant provides a universal means to characterize materials.

Importantly, the present study also shows that the devices’ dielectric response is dramatically

modified by functionalizing their surfaces using silanes. This is significant since surface

silanization is well known to be robust (even in harsh environments), compared with means to

functionalize metals typically used with nanogap elelctrodes for example. Also, silanization is well

developed due to wide spread use in chromatography as a means for tuning surface – analyte

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interactions. These results point to silicon-based, nanogap electrode devices that are

microfabricated and silane-functionalized as a target of opportunity for dielectric constant

spectroscopy - both for fundamental studies of nanoscale materials and of surfaces and for

applications to surface-tunable and selective sensing.

2.4 Experimental section

2.4.1 Impedance spectroscopy instrumentation.

The impedance analysis electronics includes a lock-in amplifier and a voltage follower. The lock-

in amplifier (Stanford Research System, model SR830 DSP) is controlled by LabVIEW software

via an RS232 cable. The voltage follower is made in house using IC model BUF634 (Texas

Instruments).

2.4.2 Nanogap device cell compartment.

To avoid current flow between contact electrodes of nanogap devices via solvent/salt solution

pathways, a custom built cell is used and consists of three parts: a PTFE base on which the nanogap

device is placed, a PTFE gasket that surrounds the aluminum part of the nanogap device surface,

and a PTFE cover with a well (id≈ 6 mm) that is filled with test solvents. The two PTFE parts are

clamped to each other via 3 screws. Two rectangular brass plates (2 mm x 6 mm) are incorporated

at the bottom of the PTFE cover to contact wires to the gold electrodes.

2.4.3 SEM imaging

Before SEM imaging, nanogap device sare rinsed with water and then propanol, and dried under

ambient conditions. The devices are attached to a similarly rinsed stainless steel sample holder

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using carbon paste. SEM images are obtained using a QUANTA FEG 250 ESEM (FEI) using a 10

kV acceleration voltage.

2.4.4 Etching procedure

All the nanogap devices are etched to remove silicon oxide using an HF-NH4F solution (1:5 v/v).

This solution is prepared using an HF solution with 40% w/w concentration and a saturated NH4F

solution. Nanogap devices are pre-rinsed with deionized water, methanol and then toluene. Next,

they are immersed in the HF-NH4F solution for 21 seconds. Immediately after etching, the devices

are rinsed with a large volume of DI water. Finally, they are left under ambient conditions for a

few days to grow a native oxide layer.

2.4.5 Silanization procedure

To functionalize the surfaces of nanogap devices, the devices are immersed in a 5% (v/v) solution

of dimethyldichlorosilane (DMDCS) in toluene for 30 minutes, and then immediately washed with

methanol and toluene. This procedure is repeated once. The devices are then washed with DI water

and left under ambient conditions for 24 hours and used for experiments.

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2.5 Results and discussion

2.5.1 Nanogap device structure

A

B

C

Figure 2. 1(A) A schematic illustration, (B) a photograph, and (C) SEM image of the nanogap device.

Figure 2.1A, 1B, and 1C show a 3D schematic, a photograph and a scanning electron microscope

(SEM) image of the nanogap device, respectively. The device is approximately 1.4 cm x 1.0 cm in

area and consists of 2 gold contacts and 3 different layers (Figure 2.1A and B). The top layer is an

aluminum electrode with circular apertures around 4 µm in diameter and 2 µm in spacing (see

Figure 2.1C). There are approximately 1.8 million apertures per device. The middle spacer layer

consists of a 40 nm thick thermally grown silicon oxide. The bottom layer is a silicon

Au(Al)

Si-SiO2

substrate

Al

Au(Si)

SiO2 (thermally grown)

SiO2 (native oxide)

Si

Al

Nanogap (annular space)

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electrode/substrate. When the device is immersed in an HF-NH4F solution for 21 seconds, wells

are etched in the silicon oxide via the apertures in the aluminum layer. This etching duration

generates wells with a diameter of ~4 µm (the size of the aperture) plus ~50 nm due to undercutting

that extends the wells under the aluminum layer.[124] As a result, an annular nanogap encircles

each well (Figure 2.1A). The depth of the wells is ~40 nm (the thickness of the silicon oxide layer).

Leaving the device in the air for days results in the formation of a native oxide layer on the silicon

surface at the bottom of the wells. The 3D schematic shows that there are at least 2 distinct regions

between the aluminum and silicon electrode layers that can provide parallel pathways for current

and that are likely to have different electronic responses: 1) a solid silicon oxide region and 2) a

series of annular nanogaps associated with the wells. These two current pathways are discussed in

more detail below in a context of an equivalent circuit model. Impedance spectroscopy is used to

explore the electronic response of the devices, and in particular to search for signatures of the

nanogaps. The latter are of particular interest as they are accessible to target analytes.

2.5.2 Impedance spectroscopy instrumentation

The impedance of the devices is studied using the circuit shown in Figure 2.2. A gain resistor

(resistance RG) is combined in series with a nanogap device (impedance Z) to form a voltage

divider.

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Figure 2. 2 Schematic diagram of the circuit used for impedance spectroscopy. Vout is the applied

voltage by the lock-in amplifier, Rint is the internal resistance of the lock-in amplifier, Z stands for

any component (nanogap device or R/C combinations) which is being tested, RG is the gain resistor,

and Vin is the voltage across RG.

The applied voltage (Vout) is divided between Z and RG, and the amount dropped across RG (VG)

is given by:

VG=VoutRG(Z+RG)-1. (equation 2.3)

VG may be phase shifted with respect to Vout.

A lock-in amplifier (LIA) is used 1) to apply a sinusoidal voltage with variable frequency

(1 Hz to 105 Hz) to the device+gain resistor combination, and 2) to measure the voltage drop across

the gain resistor. A voltage follower was used to isolate the measured voltage from all other

subsequent electronics (e.g. those inside the LIA). The LIA measures the in- and out-of-phase

components of VG (VG’ and VG”, respectively), from which the phase shift, Z’ and Z” can be

calculated using Eqs. (3) and then (2). The data may be presented by plotting Z’ and Z” versus

Computer

Voltage follower

Lock-in

amplifier

Vin Vout

Z

RG

Rint

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frequency (Bode plot), Z” versus Z’ (Nyquist plot), or VG’ and VG” versus frequency. Typically,

the latter are shown as VG’ and VG” are measured by the LIA directly. In addition, this approach

permits the effects of changing RG to be seen more easily. Various known RC combinations are

used in place of Z to test the frequency response of measurement instrumentation (see

Supplementary Materials); for example, using Z=RG=10 k, the change in phase induced by the

impedance instrumentation from 1 Hz to 105 Hz is found to be less than a few degrees.

A

B

C

D

Figure 2.3 (continues to the next page)

0

10

20

30

40

50

60

0 20 40 60 80 100 120

-Z

" -

k Ω

Z' - k Ω

0

10

20

30

40

50

60

0 20 40 60 80 100 120

-Z

" -

k Ω

Z' - k Ω

0

10

20

30

40

50

1 10 100 1000 10000 100000

VG

' -

mV

Frequency - Hz

0

10

20

30

40

50

1 10 100 1000 10000 100000

VG

' -

mV

Frequency - Hz

RG =49.4 kΩ

RG =10.0 kΩ

RG =5.03 kΩ

RG =999 Ω

RG =200 Ω

RG =49.4 kΩ

RG =10.0 kΩ

RG =5.03 kΩ

RG =999 Ω

RG =200 Ω

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E

F

Figure 2. 3 continued. Measured impedance response of the nanogap device (symbols) and calculated

equivalent circuit response (solid lines) as a function of frequency. Vout is 50 mV (rms) for all the

experiments. five different RG are used: 49.4 kΩ ( ), 10.0 kΩ ( ), 5.03 kΩ ( ), 999 Ω( ), 200

Ω( ). (a,b) Niquist plots for the device in air and propanol (respectively) based on data for RG=10.0

kΩ. (c,d) Real parts of the VG(rms values) versus frequency for the device in air and propanol

(respectively). (e,f) Imaginary parts of the VG(rms values) versus frequency for the device in air

and propanol (respectively).

2.5.3 Frequency response and equivalent circuit for the nanogap device

For the first experiments, the nanogap device is exposed to air and propanol (see Figure 2.3, left

and right columns, respectively). Figure 2.3 (A and B) show the resulting Nyquist plots for the

device (RG = 10 k); figure 2.3C-F show the in- and out-of-phase voltage drops vs. frequency

across RG (five different RG values).The Nyquist plot measured in air (Figure 2.3A) exhibits one

semicircle, and of particular interest, the one measured in propanol (Figure 2.3B) exhibits two

overlapping semicircles. Similarly, while all the in-phase responses exhibit at least one step and

the out-of-phase responses at least one peak, the in-phase data for propanol (Figure 2.3D) exhibit

two steps for some RG values, and out-

0

5

10

15

20

25

1 10 100 1000 10000 100000

VG

" -

mV

Frequency - Hz

0

5

10

15

20

25

1 10 100 1000 10000 100000

VG

"-

mV

Frequency - Hz

RG =49.4 kΩ

RG =10.0 kΩ

RG =5.03 kΩ

RG =999 Ω

RG =200 Ω

RG =49.4 kΩ

RG =10.0 kΩ

RG =5.03 kΩ

RG =999 Ω

RG =200 Ω

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Figure 2. 4 Equivalent circuit model for the nanogap device. Rs1 and C1 are attributed to the annular

nanogaps; Rs2 and C2to the bottom electrode/oxide/top electrode region; and Rp to leakage

current.

of-phase data (Figure 2.3F) exhibit two overlapping peaks. These observations are consistent with

a presence of at least two current pathways in parallel, each with a different frequency response (or

RC time constant).

Another noteworthy feature of the data is that VG’ starts from a non-zero value at low frequencies

where capacitor impedances are large, implying there should be in addition a third, parallel

pathway that is purely resistive. In view of the nanoelectrode architecture, the simplest equivalent

circuit which can describe this behavior of the device is shown in Figure 2.4 and has impedance

given by:

Z-1=(Rs1+1/iωC1)-1+(Rs2+1/iωC2)

-1+(Rp)-1, (equation 2.4)

where the resistors Rs1 and Rs2 are each in series with capacitors C1 and C2, respectively, and these

two RC series combinations are in parallel with each other and with resistor Rp.Assuming this

model, the response of the nanogap device should be effectively describable by 5 parameters which

correlate with device architecture: Rp is attributed to leakage current between the electrodes (e.g.

C2 Rs2

C1 Rs1

Rp

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due to defects in the oxide layer), Rs2 and C2 are attributed to the bottom silicon electrode/oxide/top

aluminum+gold electrode region, and Rs1 and C1 are attributed to the annular nanogaps. Rs2is

attributed to the overall contact resistances, and Rs1 to the native oxide layer and/or the aluminum

oxide layer resistances inside the nanogaps (as discussed further below).The resolution of only one

step/peak in the data for air for all RG and the observation of two steps/peaks in the data for propanol

for some RG can be caused by a difference in dielectric constants of these two solvents, namely ~1

for air and ~20 for propanol, and the resulting difference in nanogap capacitances. In view of the

voltage divider effect, the equivalent circuit model indicates that the frequencies at which the

steps/peaks occur are also strongly influenced by RG, underscoring the importance of varying RG.

Varying RG is also useful in determining the various R and C values in the equivalent circuit model

for the device- discussed further below.

The data in Figure 2.3 can be used to estimate the various parameters in this model. The

results are shown in Table 2.1. Figure 2.3 (solid lines) shows fits to the data obtained using these

values, the values of RG shown in the figure, and equations (3) and (4). The fits compare favorably

with the data for both air and propanol. It is noteworthy that there is a less than 10% difference

between all the model parameters for the device itself in propanol vs. air, except for capacitance

C1 which can be associated with the nanogap. RP and Rs2/C2, which we associate with leakage

current between electrodes and with the gold+aluminum/silicon oxide/silicon region, are device

parameters expected to have similar values independent of the medium to which the nanogap

device is exposed. Rs1 values for air and propanol are similar too, suggesting that it may arise due

to a contact

Table 2. 1 Equivalent circuit model parameters for the nanogap device in air and propanol.

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Medium Rp (kΩ) Rs2 (Ω) Rs1 (kΩ) C2 (nF) C1 (nF)

Air 120 47 18 39 5.3

Propanol 115 47 18 42 149

resistance inherent to the nanogap device. The value of C1 is significantly different for air vs.

propanol: C1,propanol/C1,air≈28. The ratio of the dielectric constants of propanol and air is about 20,

indicating that the nanogap device is indeed sensitive to the dielectric constant of the medium to

which it is exposed.

One of the utilities of the equivalent circuit model is that it straightforwardly points to an effective

method for targeting the nanogap’s sensitivity.

At low frequencies, the impedances of C1 and C2 dominate the impedances of the resistors Rs1 and

Rs2 respectively. As a result, equation (4) simplifies:

Z-1≈(iωC1)+(iωC2)+(Rp)-1, (equation 2.5)

Plotting the imaginary part of Z-1 versus angular frequency is then expected to yield a straight line

with slope (C1+C2). Figure 2.5 shows the corresponding plots obtained using the nanogap data for

air and propanol and using RG=10.0 kΩ. The coefficients of determination

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Figure 2. 5 Imaginary part of the admittance (Z-1)'' versus ω, for frequencies from 2 Hz to 10 Hz, for

a nanogap device in air and propanol (based on data shown in figure 2.4 C and D for RG=10.0 KΩ).

(R2) indicate excellent linearity of these graphs. These results suggest that the nanogap device can

effectively be used as a dielectric constant sensor in this frequency regime.

2.5.4 Sensitivity of the nanogap device to the dielectric constants of various media

To explore the nanogap device’s sensitivity to the solvent’s dielectric constant and to test the

equivalent circuit model’s ability to describe the device’s response, the device was exposed to

various solvents with a wide range of dielectric constants, and VG’ and VG’’ were measured as a

function of frequency (see Figure 2.6).

Slope = 0.0443

R² = 1.000

Slope = 0.191

R² = 0.999

0

2

4

6

8

10

12

14

0 10 20 30 40 50 60 70

(Z-1

)" -

µ S

ω - rad/s

Nanogap device in propanol

Nanogap device in air

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A

B

Figure 2. 6 Experimental data (symbols) and simulations (solid lines)for a nanogap device in

varioussolvents. For all the measurements the applied voltage was 10 mV (rms) and RG was 10.0

kΩ. (a) Real and (b) imaginary parts of the voltage (rms values) versus frequency. For clarity, the

various plots are offset by 1.5 mV.DMSO and DEEther stand for Dimethyl sulfoxide and Diethyl

ether, respectively.

As the dielectric constant increases from ε~1 for air to ε~109 for formamide,[125] VG’ initially

exhibits one step and then eventually exhibits two steps (see figure 2.6A), and VG” initially one

peak and then eventually two peaks (see figure 2.6B). These changes can be attributed to increasing

nanogap capacitance with increasing dielectric constant. The equivalent circuit’s impedance given

by equation (4) is used to generate all 18 model curves shown in figures 2.6 A and B (solid lines).

Device parameters are expected to be approximately independent of the media to which the device

is exposed. As expected, the following device parameters provided satisfactory fits in all cases:

Rp=105-120kΩ, Rs2=44-47 Ω, and C2=39-42 nF. Note that the capacitance associated with the

silicon oxide current pathway (C2) can be estimated by taking the area of the electrodes as 61 mm2,

0

5

10

15

20

25

1 100 10000

VG

' -

mV

Frequency - Hz

Formamide

DMSO

Acetonitrile

Methanol

Propanol

Pyridine

DEEther

Toluene

Air

0

2

4

6

8

10

12

14

16

1 100 10000

VG

" -

mV

Frequency - Hz

Formamide

DMSO

Acetonitrile

Methanol

Propanol

Pyridine

DEEther

Toluene

Air

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60

the separation as 40 nm and the average dielectric constant for silicon oxide as 4.25.[126] Using

Eq. (9) yields 58 nF which is in reasonable agreement with values obtained by fitting (39-42 nF).

One of the causes of the difference between the estimated and fitting values of C2 may be use of

an average value for the dielectric constant of silicon oxide (reported values range from 3.81 to

5.0).[126] The values of Rs1 for simulation are again approximately constant, ranging from 17-19

kΩ. The only model parameter that changes significantly in order to obtain reasonable fits to the

data is C1, which varies from a minimum of 5.3 for air to a maximum of 149 for propanol

confirming the nanogap device’s capacitive response. The area of each annular nanogap is

estimated as 0.71 µm2 using average values of nanogap diameter (~4.5 µm) and undercutting width

(~50 nm). To calculate the expected values for C1, this estimate for area is doubled to include the

fringes. The distance between the electrodes is 40 nm. Considering the number of nanogaps

(~1.78106) and using Eq. (9), a nanogap capacitance of ~ 0.78ε nF is expected. The values of C1

used to generate model curves range between 1.1ε and 7ε nF i.e. 1.5-9 times greater than the

expected values. This discrepancy may be due to deviations of the rate of etching,[124–127]

nanogap geometry and/or electric fields from those anticipated. Also, surface effects are expected

to be important in nanogaps and should play an important role here in the dielectric response of

molecules to electric fields.

To explore the nature of the nanogap device’s sensitivity to dielectric constant, the low frequency

response of the devices where capacitances dominate the impedance is used (equation (5)). First

the imaginary values of Z-1 generated by the various solvents with different dielectric constants are

plotted as a function of frequencies (2-10 Hz) using data in figure 2.6 (graphs not shown). Then,

the slopes of those graphs, which are equal to (C1+C2), are plotted as a function of known dielectric

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constant of the media (see figure 2.7A, data for device with bare surface). The capacitances range

from a minimum of less than 45 nF in air to a maximum of more than 190 nF in propanol.

A

B

Figure 2. 7 Slope of the imaginary part of the admittance (Z-1)'' versus frequency form 2 Hz to 10 Hz

(in nF) plotted:(a) against dielectric constant of the media for a bare and a silanized nanogap

devices, and (b) against -log of [KCl] solutions; for each graph in figure b, all the slopes are

normalized with respect to the slope for -log [KCl]=5.

Although there is some correlation in figure 2.7A between measured slope and known dielectric

constants of the solvents, the graph is clearly not linear. This is likely due to interactions between

solvent molecules and the nanogap device’s relatively large surface. Such interactions may lead

to surface adsorption, image charges on the electrode surfaces, etc., which can change the response

of the molecules to electric fields at/near the surface compared to that in the bulk. This suggests a

potential of modifying the chemical structure of the surface, solvent molecule/surface interactions

and, thereby, the nanogap’s response, as discussed below.

0.00

50.00

100.00

150.00

200.00

250.00

0 50 100

Dielectric Constant

0.9

1

1.1

1.2

1.3

1.4

1.5

1 3 5 7

Slo

pe o

f (Z

-1)"

vs ω

fro

m 2

Hz

to 1

0 H

z

(No

rm

ali

zed

)

- log [KCl]

Device with bare surface

Device with silanized surface

Device 1

Device 2

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2.5.5 Surface sensitivity of Nanogaps

Since the “bare” nanogap device used to obtain data shown in Figure 2.7A had a natural oxide

surface, strong surface interactions, e.g. with polar molecules such as alcohols, can influence the

device’s dielectric response. To explore this possibility, the surface of another nanogap device is

functionalized using Dimethyldichlorosilane (DMDCS). This molecule is chosen since silanes

provide a robust means to functionalize oxides, and DMDCS provides a hydrophobic surface due

to the methyl moeity, in contrast to the original hydrophilic oxide surface. The resulting correlation

between nanogap capacitance (C1+C2) vs. dielectric constant for various solvents using the

hydrophobic nanogap device is shown in Figure 2.7A. Unlike the case for the bare device,

capacitances vs. dielectric curves for the functionalized devices exhibit a slight minimum for

toluene and diethyl ether, even though these solvents have greater dielectric constants (2.38 and

4.33, respectively) than air, for example. Significantly, the correlation between capacitance and

dielectric constant is improved for the functionalized device compared with the unfunctionalized

devices. For example, for the unfunctionalized devices, propanol yields the largest capacitance

even though propanol does not have the largest dielectric constant. In contrast, for the

functionalized devices, the capacitance for formamide is found to be the largest, and formamide

indeed has the largest dielectric constant. The ability to alter the capacitive response of the

nanogaps devices by modifying the surface via silanization points to a significant potential of this

architecture to serve as a sensor platform. By grafting recognition elements onto the surface of this

micro-fabricated device, it may be possible to confer selectivity and/or specificity for target species

of interest.[104,113,128] This potentially could be very useful for sensing particular biomolecules,

ions, and hazardous compounds. Combined with impedance spectroscopy, dielectric sensing using

surface modified nanogap devices offer a number of benefits. They enable working in both aqueous

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and non-aqueous media and designing of recognition elements for selective interaction without

need for labels (electrochemical, fluorescent or other groups).[128,129]

2.5.6 Probing nanogaps by means of double layer thickness

To explore further the response of the nanogap device to nanoscale phenomena, the devices are

exposed to KCl solutions with a wide range of concentrations. Previous studies have shown that

the thickness of double layer changes from hundreds of nanometers at concentrations below 10-7

mol/L to a few nanometers at concentrations above 10-1 mol/L.[130,131] The capacitive response

of two unfunctionalized devices is explored using aqueous KCl solutions with concentrations

ranging from10-7 to 10-2 mol/L (see Figure 2.7B). The data show that capacitance increases with

concentration at high concentrations. Also, there is a minimum at -log [KCl]≈5. Reference 40

reports that the thickness of the double layer for this KCl concentration is about 96 nm and is 30.5

nm for 10-4mol/L. In other words, the double layer crosses 40 nm (the size of nanogap device in

the present study) between –log [KCl] of 4 and 5. As concentration increases from -log [KCl] of 4

to 2, the double layer lies within the nanogap size, and its thickness decreases. Correspondingly,

the capacitance increases significantly. At lower concentrations, the double layer thickness is larger

than the nanogap. As concentration decreases from -log [KCl] of 5 to 7, capacitance increases

slightly. This slight growth may be due to changes in dielectric constant of the solution. As the

concentration of ions decreases, fewer water molecules are required to solvate ions, more water

molecules contribute to the polarizability of the solution, and the dielectric constant increases.

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2.6 Conclusion

A microfabricated, layered architecture with nanowells and nanogaps is characterized using

impedance spectroscopy. Electronics based on a voltage divider configuration with a gain resistor

in series with the nanogap device and lock-in amplifier are used. Guided by the device’s

architecture and impedance response in various solvents and using various gain resistors, an

equivalent circuit for the device is developed. The model despite its simplicity can simulate all of

the in- and out-of-phase response data over 5 decades of frequency. Further, the model indicates -

and impedance response of the device confirm - that the nanogap device is sensitive to the dielectric

constant of media filling the nanogaps. By varying the nanogap device’s surface functionality using

silanization, the important role of the nanogap surface on device response is also confirmed.

Sensitivity of the nanogap device to nanoscale phenomena is further confirmed using salt solutions

with varying concentrations and correspondingly varying double layer capacitance. These results

are significant as the success of the model and its correlation to device architecture suggest

applicability to other nanogap devices as well. In addition, results of the present study demonstrate

that combining this microfabricated, silicon-based nanogap architecture, surface recognition

elements provided by silanization and impedance spectroscopy enables a robust and tunable

platform for dielectric constant spectroscopy at the nanoscale and of surfaces. Potential

applications range from fundamental studies to selective and/or specific dielectric constant sensors

for a variety of target species of interest.

2.7 Acknowledgments

This work was supported by funding from the Natural Sciences and Research Council for Canada

and MITACS.

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Chapter 3

3. Revisiting the nitrite reductase activity of hemoglobin with differential

pulse voltammetry

3.1 Connecting text

Haemoglobin is a conjugated protein which are responsible of a variety of reactions in the living

organism concerned with oxidation-reduction and oxygen transporting mechanisms.[132]

Hemoglobin exhibits enzymatic behavior as a nitrite reductase in vascular system.[133] It converts

nitrite (NO2-)to nitric oxide (NO) by deoxygenation of this anion.[134] NO. Recent data implicate

that the activity of NO in the vascular system is regulating by hemoglobin and the erythrocyte,

which plays a fundamental role in maintaining normal vasomotor tone.[135] UV-vis spectroscopy

has been employed extensively to study the NRA of wild-type and polymerized hemoglobins [136–

139] We used electrochemical methods by immobilizing hemoglobin on the surface of electrode

using a method published by Rusling and Nassar.[53] We used CV and DPV methods to study

nitrite reductase activity of hemoglobin and proposed a complete mechanism for the

electrochemical reaction ocuur on the surface of the electrode for the first time.

This work has been published in the following article:

Fini, H.; Kerman K. Revisiting the nitrite reductase activity of hemoglobin with differential pulse

Voltammetry. Anal. Chim. Acta, https://doi.org/10.1016/j.aca.2019.12.071, Available online 31

December 2019

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Author contributions:

All experiments were designed and discussed by Kagan Kerman and Hamid Fini. Hamid Fini

performed all the experiments. Qusai Hasan helped with some sample and electrode preparations.

Hamid Fini and Kagan Kerman discussed the results and wrote the paper.

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3.2 Abstract

Nitric oxide (NO) is an omnipresent signalling molecule in all vertebrates. NO modulates blood

flow and neural activity. Nitrite anion is one of the most important sources of NO. Nitrite is reduced

to NO by various physiological mechanisms including reduction by hemoglobin in vascular system.

In this study, nitrite reductase activity (NRA) of hemoglobin is reported using cyclic voltammetry

(CV) and differential pulse voltammetry (DPV) in a wide potential window from +0.3 V to -1.3 V

(vs. Ag/AgCl). To the best of our knowledge, a detailed look into NRA of hemoglobin is proposed

here for the first time. Our results indicated two different regimes for reduction of nitrite by

hemoglobin in its Fe(II) and Fe(I) states. Both reactions showed a reversible behaviour in the time

scale of the experiments. The first reduction displayed a normal redox behaviour, while the latter

one had the characteristics of a catalytic electro-reduction/oxidation. The reduction in Fe(II) state

was selected as a tool for comparing the NRA of hemoglobin (Hb) and hemoglobin-S (Hb-S)

under native-like conditions in a didodecyldimethyl ammonium bromide (DDAB) liquid crystal

film. These investigations lay the prospects and guidelines for understanding the direct

electrochemistry of hemoglobin utilizing a simplified mediator-free platform.

3.3 Introduction

Nitric oxide (NO) as an endothelium-derived relaxing factor (EDRF) has important

biological roles [140,141]. NO is a vital intercellular messenger that is produced in endothelial

cells, which line blood vessels. NO also mediates the signalling cascades in adjoining smooth-

muscle cells to affect the regulation of blood pressure, blood flow and oxygen delivery [142–144].

The major bioavailable pool of NO is nitrite anion in vascular system, provided that there is a

physiological mechanism which can reduce nitrite to NO [145–149]. Hemoglobin plays this role

in the vascular system by demonstrating a nitrite reductase activity (NRA) [133,138,139,150,151].

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It has been shown that a redox switch in the hemoglobin-α regulates NO diffusion in blood vessels.

Huang et al. [138] showed that, in solution, the reaction between nitrite and deoxyhemoglobin

([Hb-FeII]2+) generated methemoglobin ([Hb-FeIII]3+) and NO, which reacted with another

deoxyhemoglobin to produce nitrosylhemoglobin ([Hb-FeII-NO]2+) [138]. The kinetics profile of

the reactions exhibited autocatalytic behaviour. The stoichiometry of the reaction was 1:1 ([Hb-

FeIII]3+ to [Hb-FeII-NO]2+) under strictly anaerobic conditions, and it varied significantly in the

presence of trace oxygen contamination. Nitrosylhemoglobin may undergo further reactions in

physiological conditions [152].

UV-vis spectroscopy has been employed extensively to study the NRA of wild-type and

polymerized hemoglobins [136–139]. While the nitrite reducing ability of hemoglobin was used to

design novel electrochemical sensors for nitrite, [153–155] some efforts have also been made to

investigate the mechanism of nitrite reduction by hemoglobin using electrochemical methods

[156,157]. The challenge for electrochemical studies is to keep the behaviour of hemoglobin

consistent with its physiological functions either when it is analyzed in solution or immobilized at

an electrode surface [158]. Rusling and Nassar [53] showed that the function of myoglobin was

significantly increased, when it was immobilized in a liquid crystal film of didodecyldimethyl

ammonium bromide (DDAB) on pyrolytic graphite electrode. Later, Nassar et al. [159] showed

that the constant rates for catalytic reduction of some organohalyde pollutants by myoglobin-

DDAB modified electrodes was about 1000-fold larger compared to the ones obtained by using an

aqueous solution of myoglobin. It is not clear whether the heme protein preserves its native

structure in DDAB film. de Groot et al. [160–162] showed that DDAB most likely induces the

release of the heme group from myoglobin by means of different analytical approaches. On the

other hand, Guto and Rusling [163] used electrochemistry, UV-Vis and CD techniques and showed

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that myoglobin as a heme-protein retains iron heme and near-native conformation in DDAB films

in solutions with pH from 5 to 7 in the presence of NaBr. The influence of solution-phase HNO2

decomposition on the electrocatalytic nitrite reduction was reported by Duca et al. [164] using a

hemin-pyrolitic graphite electrode and concluded that nitrite reductases dissociate nitrite at neutral

pH. Mimica et al. [156] used the same DDAB film-based strategy for the immobilization of

hemoglobin on a glassy carbon electrode (GCE) surface to study the NRA and displayed that

hemoglobin had a similar behaviour when immobilized in a DDAB film. Throughout these studies,

many questions remained unanswered about the mechanism of the reduction of nitrite by

hemoglobin. To the best of our knowledge, all the prior literature regarding the NRA of hemoglobin

reported the catalytic reduction of nitrite mediated by hemoglobin in Fe(I) state ([Hb-FeI]1+) and/or

an irreversible peak for reduction of nitrite by hemoglobin in Fe(II) state ([Hb-FeII]2+) [155–

158,165–170]. Moreover, the oxidation of nitric oxide back to nitrite has not been reported in

electrochemical studies yet. The oxidation of NO was observed in physiological conditions,

although the final product was reported as nitrate which is a more stable species compared to nitrite

[171,172]. Here, we are reporting a detailed study to determine NRA mechanism using cyclic

voltammetry (CV) and differential pulse voltammetry (DPV). We have adopted the immobilization

of hemoglobin in DDAB liquid crystal films prepared from vesicle dispersions at pH 5 to 7 as

described by Guto and Rusling [53], to perform our studies in neutral buffers containing NaBr

under similar conditions. To compare our results with previously reported methods, human

Hemoglobin (Hb) and Sickle Cell hemoglobin (Hb-S) were utilized [150]. To the best of our

knowledge, this is the first time that the mechanism of electrochemical reduction of nitrite by

hemoglobin is investigated thoroughly.

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3.4 Experimental

3.4.1 Materials

Lyophilized human hemoglobin (Hb), lyophilized sickle cell hemoglobin (Hb-S), both of

biological grade and didodecyldimethyl ammonium bromide (DDAB), and all other reagents were

of analytical grade obtained from Sigma-Aldrich (Oakville, ON). All solutions were prepared with

deionized (DI) water obtained with a Millipore system (Woodbine, ON) after filtration with 0.4

µm filters. Nitrogen gas (99.999% purity) for electrochemical experiments was purchased from

Air Liquide (Mississauga, ON).

3.4.2 Instruments

All electrochemical tests were performed using Autolab Potentiostat/Galvanostat (PGSTAT 302N,

Metrohm AG, Herisau, Switzerland) in connection with NOVA software (NOVA 2.1.2, Metrohm

AG, Herisau, Switzerland). Transmission electron microscopy (TEM) images were obtained using

a Hitachi 7500 (Hitachi Ltd., Chiyoda, Tokyo, Japan) and were processed by iTEM software

(iTEM 5.2, Hitachi Ltd., Chiyoda, Tokyo, Japan).

3.4.3 Electrode modification

Glassy carbon electrodes (GCEs) (3.0 mm diameter) were purchased from CH instruments Inc.

(Austin, TX). To prepare the modified electrodes, GCEs were first polished with alumina powder

(1, 0.3 and 0.05 µm) and pretreated as described in literature [173]. DDAB solution was prepared

in DI water, and 0.5 mM hemoglobin was prepared in 10 mM acetate buffer (pH 5.6). DDAB

solution was sonicated for at least 4 h and mixed with the hemoglobin solution at an equivalent

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ratio [30]. Then, an aliquot (70 µL) of the desired solution (DDAB or Hb-DDAB) was transferred

onto the surface of GCEs. In order to immobilize the DDAB or Hb-DDAB complexes on GCE

surfaces, the modified electrodes were kept overnight in a closed chamber at room temperature.

3.4.4 TEM

Carbon coated grid (300 mesh) obtained from Electron Microscopy Science (Hatfield, PA). An

aliquot of desired solution (DDAB, Hb, or Hb/DDAB) was transferred onto the carbon coated grids

and allowed to dry at room temperature for a few minutes, and then used for TEM imaging. Only

for DDAB solution, a contrast agent (uranyl acetate) was used.

3.4.5 Electrochemical analysis

All solutions for electrochemical analysis were purged with nitrogen gas for at least 10 min prior

to the measurements, and for 3 min between the measurements. During the purging process,

solutions were stirred while the modified electrode was kept out of the solution. All electrodes

were placed in a sealed cell compartment (663 VA Stand, Metrohm AG, Herisau, Switzerland)

with stirring and purge on/off capabilities that were controlled by the potentiostat. Cyclic

voltammetry (CV) and differential pulse voltammetry (DPV) were performed against a double-

junction Ag/AgCl reference electrode (Metrohm AG, Herisau, Switzerland). CVs were performed

from +0.3 V to -1.3 V (unless otherwise stated) at desired scan rates. DPVs were performed from

+0.3 V to -1.3 V (unless otherwise stated) at a step potential of 5 mV with an interval of 0.3 s and

a modulation amplitude of 50 mV. All experiments were performed at room temperature.

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3.5 Results and discussion

A B C

Figure 3. 1 TEM images of 10 mM DDAB solution in DI water (A), 0.5 mM Hb in 10 mM acetate

buffer (pH 5.6) (B), and a 1:1 mixture of DDAB with Hb as described in the experimental section

(C); a solution of uranyl acetate was used only for (A) to obtain a sharp contrast (all scale bars are

5 µm).

A

B

Figure 3. 2 Cyclic voltammograms of bare GCE (solid black), DDAB-modified GCE (dashed gray)

and Hb/DDAB-modified GCE (solid red) in 100 mM phosphate buffer solution (PBS, pH 7.4) with

50 mM NaBr in the absence (A) and presence (B) of 8 mM sodium nitrite at 100 mV s-1.

-2

0

2

-1.3 -0.5 0.3

I (µ

A)

E (V vs. Ag/AgCl)

Bare GCE

DDAB-GCE

Hb/DDAB-GCE

-5

-2

1

-1.3 -0.5 0.3

I (µ

A)

E (V vs. Ag/AgCl)

Bare GCE

DDAB-GCE

Hb/DDAB-GCE

Bcat

Ban

Acat

Can

Ccat

Aan

Acat

Can

Ccat

Aan

5 µm 5 µm 5 µm

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Figure 3.1-A shows a TEM image of DDAB vesicles in water. The vesicles had various diameters

up to over 5 µm. Hbs (irregular dark spots) were randomly spread in the buffer solution as shown in

Figure 3.1-B. For Hb/DDAB solution (Figure 3.1-C), the dark spherical vesicles showed that Hbs

were mostly accumulated inside the DDAB vesicles.

As shown in Figure 3.2 (A and B), for the bare GCE in PBS, either in the absence or presence of

nitrite, CVs did not show any detectable peaks in the wide potential window between -1.3 V and

+0.3 V. A similar result was observed for the DDAB-modified GCE. Our observation showed that

the capacitive current of DDAB modified GCEs are smaller than that of the bare GCEs. We provided

some explanation for the effect of dielectric constant of DDAB and aqueous solution and the

thickness of the DDAB film and diffusion layer on the capacitive current in supplementary

information (section 1). In the applied potential window, Hb/DDAB modified GCE showed two

redox couples (Figure 3.2-A) in the absence of nitrite ions. It has been already shown that these

peaks are correlated to the oxidation and reduction reaction of iron centers in Hb: Fe(I)/Fe(II) (couple

A) and Fe(II)/Fe(III) (couple C) [156,159,174,175]. In the presence of nitrite ions, a new redox

couple appeared (couple B in Figure 3.2-B). The presence of peak Bred was reported by Mimica et

al. [156] but not completely investigated, and to the best of our knowledge has not been studied yet.

In addition, they referred to this peak as an irreversible peak that might be related to a multi-

electronic catalytic electroreduction of nitrite. However, our research showed a quasi-reversible

redox couple, when DPV was applied. The presence of nitrite ion, also, caused an increase in the

peak current of couple A showing that a catalytic reaction (in addition to a direct reduction) took

place in the presence of nitrite. We varied the scan rates from 30 mV/s to 300 mV/s for Hb/DDAB-

modified GCE in the absence of nitrite (Figure 3.S1-A). Plotting the anodic and cathodic peak

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current values vs the square root of scan rates (Figure 3.S1-B) displayed linear relationship for

couples A and C (0.999>R2>0.993). These results showed that the current for both peaks were

diffusion-controlled within this range of scan rates. This also implied that Hb molecules diffused

through the DDAB film to reach the GCE surface for electron transfer. A fast diffusion of Mb

through the DDAB layers has been reported in a work performed by Boussaad et al. [176] They

showed when the potential is raised (to around 0 V), the DDAB film transforms into a liquid-like

phase which could be responsible for the fast diffusion of Mb through the DDAB layers. Also, a

Dahms-Ruff diffusion type and/or an electron hoping mechanism may be contribute to the electron

transfer mechanism [177–179]. We performed the same scan rate dependence study for couple B

(Figure 3.S1-C). All peak potentials in CV scans for different pHs are summarized in Table 3.S1.

We observed that while the potential of reduction peak (Bred) showed a linear change vs the square

root of scan rates (R2=0.998), the potential of oxidation peak (Ban) remained relatively unchanged

as the scan rates changed (Figure 3.S1-C). This behavior could be a sign of an oxidation reaction

(Ban) with two electrons where the individual peaks were merged while the second occurring reaction

was less favorable compared to the first occurring one [180]. On the other hand, for the reduction

scan (Bred), the second reaction would be more favorable, and the peak would have shown the

characteristics of a single-step two-electron transfer. To calculate the number of electrons transferred

for each peak, the data collected in CVs were utilized. It was reported that the number of electrons

could be calculated using CV by the following equation at a constant pH [180,181].

|𝐸𝑝 − 𝐸𝑝/2| = 2.218 𝑅𝑇/𝑛𝐹 = 57/𝑛 𝑚𝑉 𝑎𝑡 25𝑜𝐶 Eq. 1

where Ep, Ep/2, R, T, n and F are peak potential of CV, the potential at which half the peak current is

observed, the universal gas constant, absolute temperature, number of electron(s) transferred and

Faraday constant, respectively. This equation was applied for well-known electrochemical reactions

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75

([Fe(CN)6]3-/4- and [Ru(NH3)6]

3+/2+ couples) and our results were in agreement with one-electron

reaction for both couples.

It was reported that the n calculated by Eq. 1 works for reversible electrochemical reactions, so for

the quasi-reversible ones the calculated n could be found smaller than the real n because of the peak

broadening which gives a bigger |Ep-Ep/2| [180]. Based on our CV results, couple A and C

corresponded to a one-electron reaction, while couple B was assigned to a two-electron reaction

[180,182].

CV Peak EP (mV) vs Ag/AgCl

(at 30 mV/s)

pH=7.4

Calculated n

(δ=3)

n

(rounded up)

Slope

of Ep vs pH

m

(number of

protons)

Can

-170 0.9 ± 0.2 1 57.2 1

Ccat

-270 0.7 ± 0.1 1 57.3 1

Ban

-777 1.2 ± 0.1 2 12.6 1

Bcat

-836 1.4 ± 0.2 2 29.4 1

Aan

-1007 1.0 ± 0.1 1 5.4 0

Acat

-1097 1.0 ± 0.1 1 6.2 0

Table 3. 1 The calculated number of electrons and protons that were exchanged in the redox

processes at electrode surface (depicted as redox couples A, B and C).

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The number of electrons calculated for each peak are summarized in Table 1. To investigate the

effect of pH on each peak, four pH values were selected in a narrow window of pH to avoid the

conformation change of Hb due to the solution pH [163]. The measurements were performed in the

absence and presence of nitrite ions; the results are shown in Figure 3.3 (A and B), respectively. For

a typical electrochemical reaction, Eq. 2 shows the dependence of peak potentials on pH [180,181].

𝐸𝑝 = 𝐸𝑝0 − (2.303 𝑅𝑇/𝐹) (𝑚/𝑛) 𝑝𝐻 Eq. 2

where Ep, Eo

p, F, m and n are the peak potential, the formal peak potential, Faraday constant, number

of protons and number of electrons, respectively. As shown in Figure 3.3-A, the peak potentials of

couple C decreased as the pH increased, but those of the couple A did not (in the absence of nitrite).

Figure 3.3-B shows the same behavior for these two peaks in the presence of nitrite. Similar to couple

C, the peak potentials of couple B (which is correlated to the redox reaction of nitrite) decreased

(Figure 3.3-B), but with a different slope.

A

B

-2.5

-0.5

1.5

-1.3 -0.5 0.3

I (µ

A)

E (V vs. Ag/AgCl)

pH 5.8

pH 6.6

pH 7.4

pH 8.1

pH increasing

-14

-6

2

-1.3 -0.5 0.3

I (µ

A)

E (V vs. Ag/AgCl)

pH 5.8

pH 6.6

pH 7.4

pH 8.1

pH increasingBcat

Ban

Acat

Can

Ccat

Aan

Acat

Can

Ccat

Aan

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77

Figure 3. 3 Cyclic voltammograms of Hb/DDAB-modified GCEs at various pH in the absence (A)

and presence (B) of 8 mM sodium nitrite in 100 mM PBS (pH 5.8, 6.6, 7.4 and 8.2) buffer with 50

mM NaBr at 100 mV s-1.

A

C

B

D

Figure 3. 4 Cathodic (A,C) and anodic (B,D) DPV for Hb/DDAB-modified GCEs in 100 mM PBS

(pH 5.8, 6.6, 7.4 and 8.2) buffer with 50 mM NaBr, in the absence (A, B) and presence of 8 mM

sodium nitrite (C, D).

Nassar et al. [49] studied the effect of pH on myoglobin in DDAB film. To investigate the effect of

pH on hemoglobin/DDAB modified electrode in more detail, DPV was employed from +0.3 V to -

1.3 V (vs. Ag/AgCl). To the best of our knowledge, this wide potential window has never been

-0.05

0.2

0.45

-1.3 -0.5 0.3

I (µ

A)

E (V vs. Ag/AgCl)

pH 5.8

pH 6.6

pH 7.4

pH 8.2

-0.05

0.75

1.55

-1.3 -0.5 0.3

I (µ

A)

E (V vs. Ag/AgCl)

pH 5.8

pH 6.6

pH 7.4

pH 8.2

-0.75

-0.35

0.05

-1.3 -0.5 0.3

I (µ

A)

E (V vs. Ag/AgCl)

pH 5.8

pH 6.6

pH 7.4

pH 8.2

-3.95

-1.95

0.05

-1.3 -0.5 0.3

I (µ

A)

E (V vs. Ag/AgCl)

pH 5.8

pH 6.6

pH 7.4

pH 8.2

Acat

Ccat

B

cat

Acat

Ccat

Dcat

Ban

Can

Aan

Dan

Can

Aan

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78

investigated before. By using DPV, we were able to show that couple B included a quasi-reversible

reaction (ΔEp≈51 mV at pH 7.4). The cathodic and anodic scans of DPV for the modified electrodes

in the absence and presence of nitrite ion is shown in Figure 3.4. The peak potentials for all couples

were plotted against pH values (Figure 3.S2) and the theoretical slope (58.16*m/n in mV/pH at 200C)

of the plots was used to calculate the m values for the number of protons corresponding to each

reaction. The results are summarized in Table 1. For couple C (Figure 3.4 A and B) the slopes for

both peaks were significantly close to the theoretical value for one electron-one proton reaction. The

peak potentials of couple A showed negligible changes as pH changed, which implied that there was

no proton involving in rate-determining step of the redox reaction of this couple. Couple B (Figure

3.4 C and D), which was of special interest for us -because it represents the nitrite reductase activity

(NRA) of Hb- displayed a complicated behavior. The reduction peak (Bcat) showed a slope equal to

29.4, which was interestingly in agreement with two electron-one proton electrochemical reaction.

The oxidation peak (Ban), on the other hand, showed a smaller slope compared to its reduction peak.

This evidence again implied that the second electron transfer of the oxidation reaction -which

involved the proton- could be slower than the first electron transfer, and then caused the peak to

broaden and could not behave like a one-step two-electron reaction. A similar two electron-one

proton mechanism has been reported for iron porphyrin complexes [183] and cytochrome P450

CYP119 [184]. Another interesting aspect of Figure 3.4 appeared, when we compared the peak

currents of couple A in the absence and presence of nitrite in different pH values. Increase of peak

currents (couple A) in the presence of nitrite -as pH decreased- showed that the mechanism of the

chemical reaction corresponding to this peak involved hydronium ion; while the electrochemical

part of it remained the same, as its peak potential did not change significantly.

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79

A

B

C

D

Figure 3. 5 Cathodic (A) and anodic (C) DPV for Hb/DDAB-modified GCE in 100 mM PBS (pH

7.4) with 50 mM NaBr in the presence of various concentrations of sodium nitrite; B and D show

the magnified images of the merged peaks for redox couples C and D.

-0.05

0.45

0.95

-1.3 -0.5 0.3

I (µ

A)

E (V vs. Ag/AgCl)

0 mM (Hb/DDAB-GCE)

2 mM (Hb/DDAB-GCE)

4 mM (Hb/DDAB-GCE)

6 mM (Hb/DDAB-GCE)

8 mM (Hb/DDAB-GCE)

12 mM (Hb/DDAB-GCE)

12 mM (DDAB-GCE)

12 mM (Bare GCE)

-0.01

0.04

0.09

-0.1 0.1 0.3

I (µ

A)

E (V vs. Ag/AgCl)

-2

-1

0

-1.3 -0.5 0.3

I (µ

A)

E (V vs. Ag/AgCl)

0 mM (Hb/DDAB-GCE)

2 mM (Hb/DDAB-GCE)

4 mM (Hb/DDAB-GCE)

6 mM (Hb/DDAB-GCE)

8 mM (Hb/DDAB-GCE)

12 mM (Hb/DDAB-GCE)

12 mM (DDAB-GCE)

12 mM (Bare GCE)

-0.25

-0.12

0.01

-0.5 -0.1 0.3

I (µ

A)

E (V vs. Ag/AgCl)

Ban

Can

Aan

Dan

Dan

Bcat

Acat

Ccat

Dcat

Dcat

Ccat

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80

The effect of nitrite concentration on DPV measurements is shown in Figure 3.5. Like their

appearance in the presence of nitrite, the height of DPV of couple B for both oxidative and reductive

peaks increased with elevating the nitrite concentration. Similar to elevation of hydronium

concentration, increasing of nitrite concentration led to an increase in the peak height of couple A.

This revealed that Hb in DDAB film did not only directly reduce the nitrite around -0.80 V (couple

B), but also reduced the nitrite through a chemical reaction, which occurred along with the

electrochemical reduction of its iron centers. This is a catalytic reduction, which had also been

observed before for different iron-porphyrin complexes [183,185] as well as cytochrome P450

CYP119 [184].

Based on the results presented above and in literature [154,155,166,186–188], Scheme 4.1 could be

proposed for the reactions occurred in couple C. The proton, which was involved in the reaction,

might attach to the histidine group or other possible ligands in the vicinity of iron center of Hb. This

phenomenon was found to be in agreement with other observations that showed the protonation-

deprotonation of histidine [189–192] or other possible ligands [193,194] attached to the iron centers

of Hb.

EC: [Hb-FeIII]3+ + H+ + e- [Hb(H)-FeII]2+

kEC-f

kEC-b

Scheme 4. 1 Reaction mechanism proposed for electrochemical reaction of couple C

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81

We hypothesized that the sixth coordination of iron center of Hb could be occupied by the small

nitrite ion. Considering the pKa (=3.398) of nitrite, this ligand could attach to (or detach from) the

iron center along with protonation (or deprotonation).[195–198] We depicted the resulted complex

with nitrite attaching to iron through its oxygen moiety in parallel with the literature, although

binding through the nitrogen moiety has also been reported [199,200]. This chemical step is depicted

in Scheme 4.2 as CHB. Considering the reduction scan, Bcat occurred in two steps. The first step (EB1)

was assumed as an electron transfer to the nitrite ligand along with a proton transfer and releasing a

molecule of water. The result would be a nitric oxide radical attached to the iron center. This

bimolecular reduction reaction was followed by another electron transfer reaction (EB2), which

produced a nitric oxide anion complexed to the iron in Hb. It was important to consider that the

second electrochemical reaction (EB2) was unimolecular and could reasonably occur faster compared

to EB1 (kEB1-f << kEB2-f). So, these two reactions can merge, and the overall electrochemical reaction

CHB: [Hb(H)-FeII]2+ + NO2

- + H+ [Hb(H)-FeII-ONOH]2+

EB1

: [Hb(H)-FeII-ONOH]2+ + H+ + e- [Hb(H)-FeII-ON.]2+ +H2O

EB2

: [Hb(H)-FeII-ON.]2+ + e- [Hb(H)-FeII-ON:]+

kCHB-f

kCHB-b

KEB1-f

KEB1-b

KEB2-f

KEB2-b

CHB2

: [Hb(H)-FeII-ON.]2+ [Hb(H)-FeIII-ON:]2+

EB2’

: [Hb(H)-FeIII-ON:]2+ + e- [Hb(H)-FeII-ON:]+ K

EB2’-f

KEB2’-b

Scheme 4. 2 Reaction mechanism proposed for reactions occur during electrochemical

reactions couple B

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82

would behave like a two-electron reaction. In contrast, for the oxidation path, EB1 would be the

second reaction that follows EB2 (kEB1-ox<<kEB2-ox). So, in oxidation scan, even though these two

peaks could merge, the second reaction would not be able to reach the speed of the first one and

result in a peak broadening. This has led to a different behavior for oxidation peak in comparison

with that of reduction peak. In parallel to EB2, another pathway was possible, which could be shown

as a combination of two reactions. First, a very fast intramolecular electron transfer (CHB2) would

produced the nitric oxide anion and Fe(III) [151]. Then, the second interfacial electron transfer could

occur at the iron center, which would have a highly favorable potential (it is noteworthy to mention

that this pathway would not change the peak current behavior and/or the overall mechanism).

As reported by Bhugun et al. [201,202], the iron center could reduce more to Fe(I) at potentials

around -1 V. In the absence of nitrite, the six coordination of iron could be stabilized by histidine or

other available ligands, and Fe(II) could be reduced to Fe(I) (shown as EA in Scheme 4.3). In the

presence of nitrite, some of the iron centers in Hb molecules could be occupied by nitric oxide ligand

as the product of EB2 or EB2’.

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83

These iron centers could reduce from Fe(II) to Fe(I) at a very similar potential, so the two peaks

could be overlapped. This reaction is depicted as EA’. It was very interesting that when the

concentration of nitrite increased the peak current of couple A also increased. This could be a

signature behavior of catalytic reduction of nitrite ions mediated by Fe(I) centers. This phenomenon

was reported for reduction of different chemical species by other types of Hb-modified electrodes

and other iron containing molecules [165,201–205]. The nitrite ion could attach to the free iron

centers with simultaneous protonation (CHA1). More protonation (CHA2) might lead to a nitric oxide

cation attached to the Fe(I) center (CHA3). Then, an intramolecular electron transfer (CHA4) produced

a nitric oxide radical with Fe(II) center. This radical could be reduced more to nitric oxide anion.

Another hypothesis is an electron transfer between Fe(I) and Fe(II) centers in the same Hb molecule

as “cooperativity” phenomenon which has been directly observed [206,207]. An X-ray study of Hb

structure showed that the maximum distance between iron centers in Hb molecule is less than 20 nm

EA: [Hb(H)-FeII]2+ + H+ + e- [Hb(H)-FeII]2+

kEA-f

kEA-b

kEA’-f

kEA’-b

EA’

: [Hb(H)-FeII-ON:]+ + e- [Hb(H)-FeI-ON:]

CHA1

: [Hb(H)-FeI]+ + NO2- + H+ [Hb(H)-FeI-ONOH]+

KCHA1-f

KCHA1-b

CHA2

: [Hb(H)-FeI-ONOH]+ + H+ [Hb(H)-FeII-ON.]2+ + H2O K

CHA2-f

KCHA2-b

EA”

: [Hb(H)-FeII-ON.]+ + e- [Hb(H)-FeII-ON:]+ K

EA”-f

KEA”-b

CHA3

: [Hb(H)-FeII-ON.]+ + [Hb(H)-FeI]+ [Hb(H)-FeII-ON:]2+ + [Hb(H)-FeII]2+ K

CHA3-f

KCHA3-b

Scheme 4. 3 Reaction mechanism proposed for reactions occur during electrochemical reactions couple A

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84

[199], which might make this cooperativity possible. In addition, intramolecular electron transfer

has been proven possible in this distance range. Although, electron transfer has been shown possible

for distances more than 20 nm in proteins through electron hoping [208]. It should be mentioned that

release of nitric oxide was possible after any of the following steps: EB2, EB2’, EA’, EA’’ and CHA5.

Release of nitric oxide after electroreduction of nitrite by iron porphyrin containing molecules has

been extensively described using optical techniques [133,136,137,139].

As the concentration of nitrite increased, our observations implied that release of nitric oxide was

partial in the time scale of DPV and CV scans. As the concentration of nitrite increased, a new couple

(Dcat and Dan) appeared and their current increased, while the peak current of couple C (both Ccat and

Can) decreased. A change in redox potential of Fe(II)/Fe(III) occurred when the sixth coordination

of iron was occupied by nitrite, and it shifted toward more positive potentials. A similar observation

was reported for iron porphyrins [183]. Scheme 4.4 shows the related reaction mechanism.

ED: [Hb-FeIII-ONOH]3+ + e- [Hb-FeII-ONOH]2+

KED-b

KED-f

Scheme 4. 4 Possible reactions proposed for couple D

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85

A

B

Figure 3. 6 DPV of Hb-S/DDAB-modified electrode in 100 mM PBS (pH 7.4) with 50 mM NaBr in

the presence of various concentrations of sodium nitrite (A), and a plot of peak current vs

concentration of sodium nitrite (B).

In order to confirm the applicability of our proposed mechanism to study nitrite reduction by Hb, we

compared the activity Hb and Hb-S. The slope of peak current of peak Bred (which-according to our

proposed mechanism-is assigned to the direct reduction of nitrite to nitrous oxide by hemoglobin)

was plotted against the concentration of nitrite (Figure 3.6-A). The slopes were found to be 87.6 and

103 nA/mM for Hb and Hb-S, respectively. These results showed a higher NRA (more than 17.5 %)

for Hb-S compared to the normal Hb. A higher NRA for Hb-S has been already shown by Fens et

al. [150] which was more than 21.8% for their experimental conditions. Another interesting result

coming out from our study was when we compared the oxidation scan in DPV of Hb and Hb-S in

the presence of various concentrations of nitrite. DPV was employed for both Hb and Hb-S from -

0.6 to 0.3 V (vs. Ag/AgCl) to study only peak Cox and Dox. Hb-S showed more decrease in Cox peak

-0.01

0.16

0.33

-0.6 -0.15 0.3

I (µ

A)

E (V vs. Ag/AgCl)

0 mM (HbS/DDAB-GCE)

2 mM (HbS/DDAB-GCE)

4 mM (HbS/DDAB-GCE)

6 mM (HbS/DDAB-GCE)

8 mM (HbS/DDAB-GCE)

8 mM (Bare GCE)

y = 0.0876x + 0.3372R² = 0.981

y = 0.1029x + 0.6946R² = 0.989

0

0.5

1

1.5

2

0 5 10

I peak

(µA

)

[NO2-] (mM)

Hb

Dan

Can

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86

current and instead more increase in Dox peak current compared to normal Hb (Figure 3.6-B). This

was attributed to a relatively more favorable interaction between the iron center and nitrite in Hb-S.

3.6 Conclusion

A DDAB liquid crystal film was used to immobilize different hemoglobins on the surface of glassy

carbon electrodes to study electrochemical reduction of nitrite by hemoglobin. Highly sensitive

DPV measurements in a wide potential window between +0.3 V and -1.3 V (vs. Ag/AgCl) were

shown to be beneficial for this purpose. DPV was used along with CV to understand the mechanism

of the related reactions in detail. A complete mechanism for all reactions was proposed for the first

time in this report. Nitrite was reduced by hemoglobin in two different regimes: Fe(II) and Fe(I)

states. Two different types of hemoglobins were also studied, and results showed that NRA

measurements using our electrochemical approach was in good agreement with previously

established spectroscopic methods of NRA detection. This electrochemical approach provides

rapid measurements of NRA utilizing significantly low quantity of reagents at an electrode surface.

We envisage that the described assay can be readily adapted to study the mechanisms of other types

of hemoglobins as well as novel synthetic metalloproteins.

3.7 Acknowledgments

This work was supported by the Canada Research Chair Tier-2 award to K. Kerman for

“Bioelectrochemistry of Proteins” (project no. 950-231116), the Ontario Ministry of Research and

Innovation (Project no. 35272), Discovery Grant (project no. 3655) from the Natural Sciences and

Engineering Research Council of Canada (NSERC), and the Canada Foundation for Innovation

(project no. 35272). The authors would like to acknowledge Alen Hadzovic for his great comments

and Qusai Hasan for his technical support to this project.

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87

3.8 Supplementary information

3.8.1 Discussions about the capacitive current observed in cyclic voltammograms

The capacitive current of electrodes in cyclic voltammetry depends on the capacitance of the

working electrode (given that the auxiliary electrode has a large surface compared to the working

electrode) and the scan rate.[180] The capacitance of an ideal plane capacitor can be calculated

by the following formula[209]:

𝐶 = ℇℇ˚ 𝐴/𝑑 eq. 3-S1

Where, C is the capacitance of the plane electrode, ℇ is the vacuum permittivity constant, ℇ˚ is the

dielectric constant of the material which fills between two planes of the capacitor, A is the surface

of plane electrode and d is the distance between the two planes.

In a solution and for a double-layer capacitor, we may consider the solid electrode surface as one

plane and an imaginary plane like the outer Helmholtz plane or a more distant plane embracing the

diffuse layer. Considering either of these latest planes as the second plane of the capacitor of our

electrodes, the solvent in the cell and the material by which the electrode surface is modified would

define the value for ℇ˚ . The fact that we used glassy carbon electrodes (GCEs) with similar

geometry for all experiments allows us to assume that A value remains the same.

In order to compare the capacitance of a bare GCE with a modified one, ℇ˚ and d values would be

compared with each other. The dielectric constant of DDAB solutions was reported to be 20.79 for

a 40% (wt%) solution.[210] Thus, it would be reasonable to consider the ℇ˚ value for DDAB film

much smaller than that of an aqueous solution (for bare GCE), which would make the C value

smaller for the DDAB-modified GCE. Similarly, the d value which is apparently larger for the

modified electrode (because of the thickness of the DDAB film) would make the C value smaller

compared to that of the bare GCE as could be observed in Fig. 3.2-A.

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88

A

B

C

Figure 3S. 1 Cyclic voltammograms of Hb-DDAB-modified GCEs in PBS (pH 7.4) with 8 mM nitrite

at different scan rates (mV/s) (a). Anodic and cathodic peak currents vs. square root of scan rate

for of Hb-DDAB-modified GCEs in PBS (pH 7.4) with 8 mM nitrite (b).

pH 8.1 pH 7.4 pH 6.6 pH 5.8

Peak Ep (V) SD (V) Ep (V) SD (V) Ep (V) SD (V) Ep (V) SD (V)

Aan -1.094 0.002 -1.091 0.005 -1.083 0.000 -1.086 0.004

Acat -1.032 0.003 -1.031 0.003 -1.025 0.002 -1.024 0.000

Ban -0.853 0.003 -0.853 0.003 -0.837 0.004 -0.825 0.010

Bcat -0.808 0.004 -0.802 0.002 -0.779 0.004 -0.743 0.010

Can -0.249 0.002 -0.221 0.002 -0.185 0.010 -0.126 0.021

Ccat -0.238 0.006 -0.160 0.022 -0.130 0.027 -0.097 0.011

Table 3S. 1 Peak potentials (Ep) of Hb-DDAB-modified GCEs under various pH conditions. Each

potential value is the average of five different scans. The solution was purged by nitrogen and

stirred between each experiment. All solutions contained 8 mM nitrite. Standard deviations (SD)

were calculated from five independent measurements (n=5).

-6

-5

-4

-3

-2

-1

0

1

2

3

-1.3 -0.9 -0.5 -0.1 0.3

I (µ

A)

E (V)

30

40

50

60

70

80

100

200

300

R² = 0.999

R² = 0.993

R² = 0.996

R² = 0.998-0.6

-0.3

0.0

0.3

0.6

0 10 20

I (µ

A)

ν1/2 (mV/s)1/2

A-oxC-oxC-redA-red

R² = 0.524

R² = 0.998-1.2

-0.8

-0.4

0.0

0.4

0 5 10 15 20

I (µ

A)

ν1/2 (mV/s)1/2

B-ox

B-red

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89

Figure 3S. 2 Anodic (an) and cathodic (cat) peak potentials of Hb-DDAB-modified GCEs vs pH

for all peaks. All solutions contained 8 mM nitrite.

y = -6.2168x - 1048.1

y = -12.632x - 752.37

y = -57.187x + 199.21

y = -5.4328x - 992.57

y = -29.424x - 577.87

y = -57.279x + 246.32

-1200

-1000

-800

-600

-400

-200

0

5.5 6.5 7.5 8.5

Ep (

mV

)

pH

A-anB-anC-anA-catB-catC-cat

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90

Chapter 4

4. A novel hybrid bilayer membrane as a platform for electrochemical

investigations of membrane proteins

4.1 Connecting text

Membrane proteins are parts of (or interact with) biological membranes specially cell membranes.

Studying these molecules brings a major challenge in protein biochemistry, since working outside

the natural lipid environment makes lots of difficulties because of the dependence of protein special

configuration on the molecular structure of the protein environment.[211] Amyloid-β as a class of

peptides which interact with cell membranes has been suggested to have a role in Alzheimer's

disease which is the most frequent neurodegenerative disorder, but the underlying mechanisms

remain uncertain.[212] Many different tools and methods has been employed to study these peptide

including electrochemical methods.[213,214] In this chapter we discuss a novel electrochemical

approach for membrane protein studies and show how we used our novel hybrid bilayer membrane

to study amyloid-β42 fibrillation.

This work has been submitted as an article to The Journal of Electroanalysis.

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91

Author contributions:

All experiments were designed and discussed by Hamid Fini, Kagan Kerman and Qusai Hasan.

Qusai Hasan and Hamid Fini performed all the experiments. Qusai Hasan, Hamid Fini and Kagan

Kerman discussed the results and wrote the paper.

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92

4.2 ABSTRACT

Herein, a novel hybrid bilayer membrane is introduced as a platform to study the aggregation of

amyloid-β1-42 (Aβ1-42) on glassy carbon electrode (GCE) surfaces. Both positively- (Ru(NH3)6) and

negatively-([Fe(CN)6]3-/4-) charged redox probes were used for electrochemical characterization of

modified surfaces using cyclic voltammetry (CV) and electrochemical impedance spectroscopy

(EIS). EIS results showed a decrease in charge transfer resistance (Rct) upon incubation of Aβ1-42

on the hybrid bilayer-modified surfaces. This proof-of-concept study provides a promising

electrochemical platform for designing hybrid bilayers with various physicochemical properties to

study the interaction of membrane-bound receptors and biomolecules on surfaces.

4.3 INTRODUCTION

All living cells are composed of a semi-permeable membrane, which acts as the frontier between

the cell and its environment.[34,215–217] Studies of the cell membrane have revealed that it is

mainly composed of a phospholipid bilayer with properties that are different from those of any of

its individual components.[36] Furthermore, cell membranes are considered to be a fluid mosaic

as they possess several dynamic types of proteins, carbohydrates, and glycolipids.[36,218]

Synthetic bilayer membranes made of lipids have been broadly employed as models to study

biomembranes since they were first introduced in the form of black lipid membranes by Mueller

et al. [218] in 1962. Formation of biomembrane-like bilayer structures from a simple organic

compound was introduced by Kunitake and Okahata [219] in 1977. Direct -specifically in situ-

investigation of biological membranes is a challenging topic of intense research interest. To study

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93

membrane proteins, researchers developed biomimetic membranes using various approaches

which stabilize the bilayers on a solid substrate surface. A wide variety of biomimetic membranes

have been developed in the past decade.[41,220–228] Hybrid bilayer lipid membranes (HBLMs)

are often composed of a thiolated-alkyl chain covalently attached to a gold surface, resulting in the

formation of a self-assembled monolayer, while the second layer is typically composed of a

phospholipid.[221,223,224,229] The covalent nature of the first layer of the HBLMs provides

stability against changes in pH, ionic strength and composition of buffer solutions.[230–232]

Because of their rugged formation on surfaces, HBLMs have been studied using a wide variety of

optical and electrochemical techniques.[233–238]

Electrodeposition of diazonium on surfaces has been a powerful technique in surface

chemistry.[62,86,239–242] In this study, diazonium salts have been employed to prepare a hybrid

bilayer membrane (HBM). The first and the second layers of HBM were characterized by time-of-

flight secondary ion mass spectrometry (TOF-SIMS), X-ray photoelectron spectroscopy (XPS),

cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). This platform was

then used to study the aggregation of amyloid-β1-42 (Aβ1-42), a hallmark protein in Alzheimer’s

disease (AD) using EIS in connection with positively- (Ru(NH3)6) and negatively-(([Fe(CN)6]3-/4-)

charged redox probes. One of the well-studied aspects of AD pathology has been focused on Aβ

peptides that form neurofibrillary tangles and plagues in the brains of AD patients.[243–246] The

soluble intermediate oligomers formed during the aggregation process are hypothesized to present

the highest neurotoxicity.[247–249] Research has demonstrated that Aβ1-42 aggerates more rapidly

than Aβ1-40 due to the additional two hydrophobic residues in Aβ1-42.[250–252] Since the

aggregation of Aβ peptides have been a topic of significant interest, there have been numerous

studies to follow this process using electrochemical techniques.[253–260] Our results

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demonstrated that long-chain HBM (lcHBM) provided a promising platform for studying Aβ1-42

aggregation toward a wide range of drug screening applications targeting the neurotoxicity of Aβ1-

42 aggregates.

4.4 EXPERIMENTAL METHODS

4.4.1 Chemicals

HBF4 (48% in H2O), 4-dodecylaniline (97%), 4-ethylaniline (98%), NaNO2 (97.0%), acetic acid

(≥99.7%), propionic acid (≥99.5%), acetonitrile (≥99.9%), tetrabutylammonium tetrafluoroborate

(>99%), dihexadecyl phosphate (DHDP), potassium hexacyanoferrate(III) K3[Fe(CN)6],

potassium hexacyanoferrate(II) trihydrate, K2[Fe(CN)6], (≥99.5%), 1,1,1,3,3,3-hexafluoro-2-

propanol (HFIP, ≥99.9%), ruthenium hexamine (Ru(NH3)63+) and phosphate buffered saline (PBS)

tablets were obtained from Sigma-Aldrich (Oakville, ON). Hydrochloric acid (Reagent grade) was

obtained from Caledon Laboratories Ltd. (Georgetown, ON). All solutions were prepared with

deionized water obtained with a Millipore system (Woodbine, ON) after filtration with 0.4 µm

filters. Nitrogen gas (99.999% purity) for electrochemical experiments was purchased from Air

Liquide (Mississauga, ON).

4.4.2 Instruments

All electrochemical tests were performed using Autolab Potentiostat/Galvanostat (PGSTAT 302N,

Metrohm AG, Herisau, Switzerland) in connection with NOVA™ software (NOVA 2.1.2,

Metrohm AG, Herisau, Switzerland). Transmission electron microscopy (TEM) images were

obtained using a Hitachi 7500 Transmission Electron Microscope equipped with an Olympus SIS

MegaView II 1.35 MB digital camera (Hitachi Ltd., Chiyoda, Tokyo, Japan) and were processed

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by iTEM™ software (iTEM 5.2, Hitachi Ltd., Chiyoda, Tokyo, Japan). Time-of-Flight secondary-

ion mass spectrometry (TOF-SIMS) measurements were performed using TOF-SIMS IV (Ion-ToF

USA Inc., NY, NY). Thermo-Scientific K-Alpha (Mississauga, ON) was used for X-ray

photoelectron spectroscopy (XPS) measurements. Agilent 6530 Q-TOF (Santa Clara, CA) mass

spectrometry (MS) and Alpha-P Fourier-transform infrared (FTIR) spectroscopy (Bruker, Billerica,

MA) were utilized for chemical characterizations of the synthesized molecules.

4.4.3 Electrode pretreatment

Glassy carbon electrodes (GCEs) (3.0 mm diameter) were purchased from CHInstruments Inc.

(Austin, TX). To prepare the modified electrodes, GCEs were first polished with alumina powder

(1, 0.3 and 0.05 µm), followed by sonication in deionized water and subsequently 95% ethanol for

30 min each. The electrode surface was then acid-activated by running a cyclic voltammogram in

1 M H2SO4 for 15 scans between -1.2 and +1.2 V (vs. Ag/AgCl) at a scan rate of 0.1 V/s. DHP and

BEHP solutions (10 mM) were prepared in deionized water.

4.4.4 Synthesis of 4-dodecylbenzenediazonium tetrafluoroborate (DDAN)

4-Dodecylbenzenediazonium was prepared from 4-dodecylaniline using procedures as reported in

literature.[261–263] Briefly, 4-dodecylanaline (0.5 g, 2 mmol) was mixed into a solution that

contained equal volumes of acetic and propionic acid (7 mL in total), to which, 2.5 mL of HBF4

was added. The solution was cooled to 6oC, to which 0.2 g of NaNO2 was slowly added. The

mixture was stirred for 1 h at 6oC and was subsequently vacuum filtered to isolate the product

which was washed with ethanol and dried. The synthesis reaction yield was measured

approximately as 85.1%. MS and FTIR spectra of the product is shown in Figures S1 and S2.

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Isolated product (yellow-orange) was stored in a sealed container over CaCl2 at 4oC until further

use.

4.4.5 Synthesis of EDAN

4-Ethyldiazonium was prepared in situ using a solution of 4-ethylaniline (2 mM) with an equimolar

sodium nitrite in 1.25 M HCl solution. This solution was purged with nitrogen gas for at least 10

min.

4.4.6 Electrode modification

For the modification of GCE surfaces with lcHBMs, GCEs were modified by the 4-dodecylbenzene

moiety by first dissolving 4-dodecylbenzene (2 mM) and tetrabutylammonium tetrafluoroborate

(10 mM) in acetonitrile. CV was run between -0.9 V and +0.6 V for 30 scans at 0.05 V/s (Figure

1), resulting in the formation of the first layer. The electrodes were taken out the solution, rinsed

with deionized water and sonicated in deionized water for 1 min. In order to adsorb the second

layer, a solution of was prepared by mixing 10 mM DHDP in deionized water along with equimolar

NaOH to assist dissolution. The solution was then sonicated for 2 h in order to form a homogenous

mixture. After which, GCEs that were formerly modified with the first layer were incubated with

100 µL of the solution overnight. For the modification of GCE surfaces with scHBMs, immediately

following the synthesis of EDAN in situ, CV was applied between 0.6 V and -1.2 V vs Ag/AgCl

for 10 cycles at 50 mV s-1 scan rate (Figure S3). The electrodes were taken out the solution, rinsed

with deionized water and sonicated in deionized water for 1 min. Electrodes modified with 4-

ethylbenzene were then incubated with a 10 mM solution of BEHP in deionized water.

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4.4.7 Electrochemical measurements

The modified GCEs (both ethyl and dodecyl bilayers) were analyzed electrochemically using

cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in either a 2.5 mM

solution of [Fe(CN)6]3-/4- with 50 Mm NaBr or a 2 mM solution of Ru(NH3)6

3+ with 50 mM NaBr.

CV measurements were performed at a scan rate of 50 mV s-1, unless otherwise stated. EIS data

were collected as Nyquist plots with an applied bias of -0.20 V and +0.25 V vs Ag/AgCl for

measurements performed using Ru(NH3)63+ and [Fe(CN)6]

3-/4-, respectively at a frequency ranging

from ranging from 100 mHz to 100 kHz.

4.4.8 Aβ1-42 aggregation studies

Aβ1-42 was obtained from AnaSpec Inc. (Fremont, CA). Aβ1-42 was first treated using HFIP as

described before.[264] Briefly, 0.5 mL of HFIP was added to 1 g of Aβ1-42 which was then

sonicated for 15 min. The solution was then stored at 4oC overnight. The solution was then

aliquoted after which the HFIP was dried under N2. Aβ1-42 films were then stored at -20oC until

they were further needed. Immediately before measurement, 1 mL of 0.01 M phosphate buffer (pH

7.4) was added to dissolve the peptide. lcHBM-modified GCEs were then incubated with 100 µL

of the peptide solution for 10 min, 24 h, and 48 h. Aβ1-42-modified lcHBM surfaces were gently

washed with deionized water immediately before EIS measurements.

4.5 RESULTS AND DISCUSSION

4.5.1 Covalent modification of GCE surfaces

To prepare the hybrid bilayer, 4-dodecylbenzenediazonium (DDAN) and 4-

ethylbenzenediazonium (EDAN) were utilized as precursors to covalently attach the alkylaryl

chains onto the GCE surfaces. As described in the Experimental section (also shown in Scheme

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S1), DDAN was synthesized, isolated and characterized using MS (Figure S1) and FT-IR (Figure

S2). EDAN was prepared in situ using the corresponding aniline salt (Scheme S2). To graft DDAN

onto GCE surfaces, thirty consecutive cycles of CV were performed at a scan rate of 0.05 V s-1

between 0.60 V and -0.90 V vs. Ag/AgCl. The corresponding chemical reaction is illustrated in

Figure 1A. Figure 1B shows the voltammograms for the first, second and the last two

voltammograms for DDAN grafting. Our investigations showed that after thirty cycles, the current

difference between the last two voltammograms displayed less than 2% difference for almost all

measurements. Cyclic voltammograms of EDAN electrodeposition are shown in Figure S3.

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Figure 4.1 Schematic diagram for the electrodeposition of DDAN on a GCE surface (A). CV of 2

mM DDAN in acetonitrile along with 10 mM of tetrabutylammonium tetrafluoroborate at a scan

rate of 0.05 V s-1 between +0.60 V and -0.90 V vs Ag/AgCl: scans 1, 2, 29 and 30 (last scan) are

shown (B).

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4.5.2 Preparation of the hybrid bilayer membranes (HBMs)

To prepare the second layer, dihexadecyl phosphate (DHP) was chosen for the electrodes with the

first layer made of DDAN. For electrodes with EDAN as the first layer, bis(2-ethylhexyl)

phosphate (BEHP) was chosen as the second layer. A solution of 10 mM DHP was prepared and

sonicated for 2 h to form vesicles prior to incubation. Figure 2A shows TEM images of these

vesicles. Subsequently, 100 µL of the prepared solution was incubated on the DDAN-modified

surfaces at room temperature overnight. The modified GCEs were then gently rinsed with

deionized water followed by a 1 min sonication of the GCE-DDAN-DHP, which is coined here as

the long-chain hybrid bilayer membrane (lcHBM) to remove the non-specifically adsorbed DHP

molecules/vesicles.

Figure 4.2 TEM images of a solution of 10 mM of DHP (A, B), and 10 mM of DHP along with 10

μM of Aβ1-42 (C, D).

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The same procedure was performed to synthesize GCE-EDAN-BEHP using a 10 mM solution of

BEHP, which is coined here as the short-chain hybrid bilayer membrane (scHBM). Figure 3 shows

an illustration of the long-chain hybrid bilayer (lcHBM). It should be noted that first and second

layers could have different defections as we hypothesized some of possible multilayers might have

been formed during electrodeposition. For future studies, the formation of multilayers can be

prevented by adding a radical scavenger such as 2,2-diphenyl-1-picrylhydrazyl in excess [33].

Research in our laboratory is in progress to verify this hypothesis.

Figure 4.3 Schematic illustration of the deposition of the biomimetic membrane on a GCE surface.

The electrodeposition of DDAN on the surface of GCE (A). The immobilization of DHP was

performed by the incubation of DHP vesicles on the surface overnight (B).

Surface characterizations of the HBMs were performed using XPS and TOF-SIMS. XPS results

show a distinctive phosphorus peak for the modification of GCE surface with DDAN-DHP (Figure

4). TOF-SIMS data for bare GCE, GCE-DDAN, and GCE-DDAN-DHP are shown in Figure 5A

and 5B. In Figure 5A, the spectra with negative polarity shows the mass of 4-dodecylbenzene

which formed the first layer of lcHBM (DDAN) for both GCE-DDAN and GCE-DDAN-DHP and

no considerable peaks for bare GCE. The spectra with positive polarity are shown in Figure 5B

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where the mass of dihexadecyl phosphate (DHP; the second layer of lcHBM) only for GCE-

DDAN-DHP and no considerable peaks for bare GCE and GCE-DDAN.

Figure 4.4 XPS spectra for the GCE-DDAN-DHP modification showing a clear distinct phosphorus

peak with an asymmetric peak envelop due to the overlapping P2p3/2 and P2p1/2 spin orbit

components.

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Figure 4.5 TOF-SIMS spectra for all three surfaces (500x500 μm2 area): bare glassy carbon

electrode (GCE), the first layer-modified electrode (GCE-DDAN) and bilayer-modified electrode

(GCE-DDAN-DHP) at negative (A) and positive (B) polarity.

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4.5.3 Electrochemistry of lcHBM- and scHBM-modified surfaces

Ruthenium hexamine ([Ru(NH3)6]3+ and ferri/ferrocyanide ([Fe(CN)6]

3-/4-, having overall positive

and negative charges, respectively, revealed the electrochemical characteristics of lcHBM- and sc-

HBM-modified surfaces. Figure 6A shows a reversible CV (blue line) for [Ru(NH3)6]3+ on a bare

GCE, while in the CV (red line) of the first layer (GCE-DDAN), [Ru(NH3)6]3+ shows no detectable

peaks under the given conditions. After incubation of the second layer (DHP), a small broadened

reductive peak of [Ru(NH3)6]3+ was observed. We attributed this phenomenon to the presence of

negatively charged phosphorous groups which facilitated the diffusion of the positively charged

[Ru(NH3)6]3+ to the surface. EIS results for the same electrode are shown in Figure 6B. The blue

graph (insertion) shows the small charge transfer resistance (Rct) of [Ru(NH3)6]3+ on a bare GCE.

The red graph shows a relatively high Rct when the electrode was modified with the first layer

(GCE-DDAN). Similar to CV, EIS for [Ru(NH3)6]3+ in the presence of a second layer (GCE-

DDAN-DHP), a decrease in Rct was observed compared to the first layer (green graph). These

behaviors were reproducible for all measurements performed in different days with new solutions

and different GCE surfaces. A similar behavior was observed for scHBM (which consists of shorter

molecules for both first (EDAN) and secondary layers (EDAN-BEHP)) and the results are shown

in Figure S4.

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Figure 4.6 Cyclic voltammograms and Nyquist plot of the bare GCE, DDAN layer (the first layer),

and DDAN-DHP bilayer. (A) and (B) show the respective CV at a scan rate of 50 mV s-1 and EIS

using [Ru(NH3)6]3+ as the positively-charged redox probe. (C) and (D) show the respective CV and

EIS spectra using [Fe(CN)6]3-/4- as the negatively-charged redox probe. In Nyquist plots, all dots

represent the experimental data and solid lines represent the simulated data with the applied bias -

0.20 V and +0.25 V vs Ag/AgCl for measurements performed using Ru(NH3)63+ and [Fe(CN)6]

3-

/4-, respectively, at a frequency from 100 mHz to 100 kHz with an amplitude of 5 mV. Insets shows

plots for bare GCE.

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[Fe(CN)6]3-/4- as a negatively charged electrochemical probe showed results in a reverse trend

compared to the positively charged [Ru(NH3)6]3+ (Figure 5C and 5D). CV (blue line) in Figure 5C

shows the reversible voltammogram of [Fe(CN)6]3-/4- on a bare GCE. While the GCE-DDAN and

GCE-DDAN-DHP showed no detectable peaks under the given conditions. EIS results showed a

relatively high Rct of [Fe(CN)6]3-/4- on a GCE-DDAN (red) while for [Fe(CN)6]

3-/4-, the presence of

the second layer (GCE-DDAN-DHP) adds to the semicircle diameter in EIS (green) which was

attributed to the repulsion of the negatively charged groups in the second layer. For scHBM, a

similar behavior was observed which is shown in Supplementary Information Figures S2C and

S2D. To simulate all EIS graphs, a modified Randles equivalent circuit (Inset of Figure 7) was

utilized with all values of the equivalent circuit elements as summarized in Table S1.

Figure 4.7 EIS of modified GCEs in the presence of [Fe(CN)6]3-/4- with EDAN layer (the first layer)

(GCE-EDAN; red circles), and bilayer (GCE-EDAN-BEHP; green circles). Results are shown

before (filled circles) and after (empty circles) stirring with the redox probe at 500 rpm for 1 min

with the applied bias of +0.25 V vs Ag/AgCl at a frequency ranging from 100 mHz to 100 kHz

with an amplitude of 5 mV.

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To investigate the mechanical stability of the bilayer, we stirred both GCE-EDAN and GCE-

EDAN-BEHP with 500 rpm for 1 min. EIS results are shown for both layers, before and after high

speed rotation (Figure 7). Values of the simulated electrical circuit elements are shown in Table

S2. Comparison of each data showed that both layers were stable after a high-speed rotation.

Further work towards electrochemical kinetic investigations using HBM-modified rotating disc

electrodes are in progress in our laboratory.

4.5.4 Interaction with Aβ1-42

Aβ1-42 was used as a model biomolecule to explore the applications of lcHBM-modified surfaces.

To study the interaction of Aβ1-42 with the lcHBM, we prepared a fresh solution of 10 µM of Aβ1-

2 in PBS (pH 7.4) and incubated an aliquot of the peptide solution (20 μL) for 10 min, 24 h and 48

h on the lcHBM-modified surfaces. After incubation, the electrodes were washed thoroughly with

PBS and then deionized water before EIS measurements (Figure 8) were taken using [Ru(NH3)6]3+

and [Fe(CN)6]3-/4- as redox probes. As shown in Figures 8A and 8B, the Rct of Aβ1-42-modified

surfaces decreased significantly after 10 min of incubation time (purple). This decrease in Rct was

attributed to the aggregation of Aβ1-42 on the bilayer creating disruption on the ordered membrane

surface that facilitated the diffusion of redox probes to the GCE surface. The disruption of neuronal

cell membranes with the oligomers and fibrils of Aβ1-42 to form pores has been previously described

to cause neurotoxicity [41-46]. The significant decrease in Rct was observed with a similar trend

for both redox probes, despite the fact that the charges of probes were opposite. This strengthened

our hypothesis that pores or disruptions in the lcHBM layer took place, which facilitated the charge

transfer between GCE surface and the redox probes. Further studies aiming to observe the

formation of Aβ-based pores on lcHBM-modified surfaces are in progress in our laboratory using

atomic force microscopy (AFM). Another noticeable trend in EIS results was that a significant

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decrease in Rct was observed after a short time (10 min) of incubation with Aβ1-42. The difference

between the Rct values obtained at 24 h and 48 h of incubation was not statistically significant

(Table S3). These observations implied that the disruption of the lcHBM layer took place at the

early stages of Aβ1-42 aggregation. In agreement with our results, Zhao et al. [265] modified a

supported bilayer lipid membrane on a gold electrode surface, which induced a significant steric

hindrance that prevented [Fe(CN)6]3-/4- from approaching the electrode. However, after incubation

of the modified electrode with Aβ peptides, a high current response of [Fe(CN)6]3-/4- can be

obtained using differential pulse voltammetry, indicating the formation of ion channels in the lipid

membrane [61].

The ability of Aβ peptides to disrupt membrane integrity have been studied by numerous research

groups in the past decade. Linberg et al. [266] reported that lipid membranes of

dioleoylphosphatidylcholine (DOPC) catalysed the fibril formation of Aβ1-42 through lipid-fibril

interactions that reinforced secondary pathways. Single molecule microscopy was applied to track

individual Aβ peptide diffusion on lipid bilayers by Chang et al. [267] reporting that trimers and

larger oligomers were immobilized on the lipid bilayer. Niu et al. [268] have reviewed the

mechanism of Aβ-membrane interactions. Khondker et al. [269] correlated the aggregation of the

hydrophobic fragment of Aβ25-35 with the hydrophobicity, fluidity and charge density of a lipid

bilayer. Kandel et al. [270] have recently studied the cholesterol-dependent membrane pore

formation of Aβ25-35 peptides. Bharadwaj et al. [271] have described the role of cell membrane

interface in modulating the production and uptake of Aβ peptides. Additional evidence was

provided by Capone et al. [272] suggesting that Aβ peptides induced ion channel-like ion flux in

cellular membranes that was independent from the postulated ability of Aβ to modulate intrinsic

ion channels or transporter proteins. Lal et al. [273] reviewed the high-resolution 3D structure of

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Aβ channels and their relevance to amyloid channel paradigm. Drolle et al. [274] utilized atomic

force microscopy (AFM) to study the molecular mechanisms of amyloid fibril formation and

toxicity in AD. Recently, it has been established that Aβ oligomers had a profound detergent-like

effect on lipid membrane bilayers as imaged by AFM and electron microscopy.[275]

Electrochemical and AFM investigations to discover small molecules that would affect the

interaction of Aβ1-42 with the lcHBM-modified surfaces are in progress in our laboratory.

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Figure 4.8 EIS of a GCE-DDAN-DHP using [Ru(NH3)6]3+ (A) and [Fe(CN)6]

3-/4- (B) as the redox

probe (green), and after 10 min (purple), 24 h (yellow) and 48 h (blue) incubation in a solution of

Aβ1-42. All dots represent the experimental data and solid lines represent the simulated data with

the applied bias of -0.20 V vs Ag/AgCl with [Ru(NH3)6]3+ (A) and +0.25 V vs Ag/AgCl with

[Fe(CN)6]3-/4- at a frequency ranging from 100 mHz to 100 kHz and a potential amplitude of 5 mV.

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4.6 CONCLUSIONS

Our preliminary results displayed the synthesis and electrodeposition of a novel hybrid bilayer

membrane on glassy carbon surfaces. The lcHBM-modified GCEs were utilized to study Aβ1-42

aggregation process upon interaction with the surface-anchored membrane. This platform can be

customized according to the purpose of the study by choosing appropriate molecules that are used

for diazonium salts for the covalently attached first layer as well as the phospholipid (or other

bipolar molecules) as the second layer. A combination of different molecules can also be used for

the two layers to adjust the affinity, thickness and other physical properties of the HBMs. We

envisage that similar EIS studies can be performed using lcHBM-modified GCES in connection

with membrane-bound biomolecules to understand their interactions with small molecules in drug

screening assays.

4.7 ACKNOWLEDGMENTS

This work was supported by the Canada Research Chair Tier-2 award to K. Kerman for

“Bioelectrochemistry of Proteins” (project no. 950-231116), the Ontario Ministry of Research and

Innovation (Project no. 35272), Discovery Grant (project no. 3655) from the Natural Sciences and

Engineering Research Council of Canada (NSERC), and the Canada Foundation for Innovation

(project no. 35272).

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4.8 Supplementary information

Scheme 4S.1 Schematic diagram for synthesis of 4-dodecylbenzenediazonium tetrafluoroborate

(DDAN).

Scheme 4S.2 Schematic diagram for in situ synthesis of 4-ethylbenzene diazonium (EDAN). As

described in the Experimental section, 2 mM of ethyl aniline was dissolved in 1.25 M HCl followed

by the addition of 1 equivalent of NaNO2.

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Figure 4S.1 Mass spectroscopic characterization of the synthesised diazonium salt (DDAN, 4-

dodecylbenzene-diazonium tetrafluoroborate).

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A

B

Figure 4S.2 IR spectra for the synthesised diazonium salt (DDAN, 4-dodecylbenzene

diazonium tetrafluoroborate) and the precursor amine (4-dodecylaniline) (B).

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Figure 4S.3 Cyclic voltammograms for the electrodeposition of 4-ethylbenzenediazonium (EDAN)

on GCE surface at a scan rate of 50 mV s-1 (refer to Scheme S2).

-13

-9

-5

-1

3

-1.2 -0.6 0 0.6

I / µ

A

E / V vs. Ag/AgCl

1st Scan

2nd Scan

10th Scan

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Figure 4S.4 Cyclic voltammograms and Nyquist plots of bare GCE, GCE-EDAN layer, and GCE-

EDAN-BEHP bilayer. (A) and (B) show the respective CV and EIS spectra using [Ru(NH3)6]3+ as

the redox probe. (C) and (D) show the respective CV and EIS spectra using [Fe(CN)6]3-/4- as the

redox probe. In Nyquist plots, all dots represent the experimental data and solid lines represent the

simulated data using [Ru(NH3)6]3+ and [Fe(CN)6]

3-/4- at an applied bias of -0.20 V and +0.25 V vs

Ag/AgCl at a frequencies range between 100 mHz and 100 kHz with a potential amplitude of 5

mV.

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Table 4S. 1 The values of simulated equivalent circuit elements of Nyquist plots shown in Figures 6B

and 6D.

Electrochemical

probe

Surface

modification Rs Rct Cdl N Cw M

[Ru(NH3)6]3+

Bare GCE 40 0.262 4.45 1 215 0.5

DDAN 232 55.6 48.2 0.946 5.81 0.415

DDAN-DHP 210 48.4 740 0.919 38.6 0.46

[Fe(CN)6]3-/4-

Bare GCE 123 0.148 4.2 0.413 260 0.525

DDAN 189 30 60.5 0.935 5.18 0.51

DDAN-DHP 188 67.3 57.2 0.943 2.86 0.473

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Table 4S.2 The values of simulated equivalent circuit elements of Nyquist plots shown in Figure 7 with

the redox probe [Ru(NH3)6]3+.

Surface

modification

Stirring

time Rs Rct Cdl N Cw M

GCE-DDAN No stirring 235 3.38 156 0.892 80.8 0.404

1 min 232 3.82 155 0.892 71.9 0.404

GCE-DDAN-

DHP (Bilayer)

No stirring 176 754 498 0.766 145 0.466

1 min 171 588 638 0.744 174 0.472

Table 4S.3 The values of simulated equivalent circuit elements of Nyquist plots shown in Figure 8.

Electrochemical

probe

Incubation

time Rs Rct Cdl N Cw M

[Ru(NH3)6]3+

0 193 1020 39.8 0.952 560 0.493

10 min 181 285 46.9 0.935 1070 0.526

24 h 173 110 58.8 0.915 1740 0.531

48 h 204 102 59.2 0.915 1930 0.495

[Fe(CN)6]3-/4-

0 196 1320 38.9 0.960 236 0.352

10 min 183 974 46.7 0.945 392 0.415

24 h 197 291 49.2 0.937 1120 0.552

48 h 204 240 48.3 0.941 1330 0.565

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Chapter 5

5. Future directions

5.1 Microfabricated, silicon devices with nanowells and nanogap electrodes

We showed that nanogap device (described in chapter 2) is sensitive to the change of capacitance

either with respect to the dielectric constant of the medium filling the gaps, or due to the change of

double layer capacitance inside the gap. As a result, this nanogap chip can be used as a platform

for different analytes in a way that they change the capacitance of the nanogaps. An interesting

group of analytes for this purpose are heave metals. A wide range of sensor has been developed for

sensing heavy metals.[276–278] A very important heavy metal which may be present in many

environmental samples as cations is mercury. According to United States Environmental Protection

Agency (EPA), the level of mercury ion in tap water should be lower than 2 ppb.[279] A selective

recognition element is necessary for making a sensor out of this nangop device.[280–282] many

different recognition elements have been used for mercury ion like protein[106], nucleic acid[283],

small molecules[284,285] macromolecules[286] and crown ethers[287–290]. A very interesting

candidate element for mercury detection by our nanogap device is could be s a recognition

consisting of a moiety as 4-(1-oxa-4,10-dithia-7-aza-cyclododec-7-yl) which is shown in Figure

5.1.A; I will use CE (crown ether) as an abbreviation for this molecule here after.

A B

Figure 5. 1 Chemical structure of 4-(1-oxa-4,10-dithia-7-aza-cyclododecane) (A) and 4-(1-oxa-4,10-dithia-7-aza-

cyclododec-7-yl) (B).

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We first wanted to test the ability of this molecule for reversible capturing of mercury ion in

aqueous solutions. So, we designed a molecule which enables us of testing this moiety as part of

a molecule grafted on quartz slides in a way that we can use UV-Vis spectroscopy for

investigation of this molecules ability before using in our capacitive nanogap device. The

molecule we targeted for synthesis is shown in Figure 5.1.B. We synthesized this molecule as

demonstrated in Scheme 5.1.

A

B

C

Scheme 5. 1 Synthesis of CE

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D

E

Scheme 5.1 continued- Synthesis of CE

Having our CE, I used (3-Aminopropyl)methyldichlorosilane to modify the quartz surface with an

amine group (Scheme 5.2 A). Then used aldol condensation to graft the CE on the surface.[291,292]

Figure 5.2-A shows spectrograms for two quartz slides: a bare slide and a slide modified with CE.

A peak around 280 nm is distinguishable for CE modified slide which is consistent with the

benzene absorption. To test the interaction of CE with Hg, I used a CE modified quartz slide and

exposed it to a solution of 10 ppb Hg2+ and then washed the slide with distilled water and dried

gently with a flow of nitrogen gas. The resulted UV-Vis spectrum showed a 20 nm blue shift in the

UV-Vis spectrum (Figure 5.2-B) which is a sign of interaction of the crown ether moiety with Hg.

Then, I washed the slide with a solution of 10 mM EDTA and washed with water and dried with

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nitrogen. This process brings the peak back to 280 nm which shows the interaction between the

crown ether and Hg is reversible. I repeated the above procedure and showed that the whole process

is repeatable. All these results show that using this synthesized molecule is a promising recognition

element which can be used for our nanogap sensor.

A

B

Scheme 5. 2 Modifying quartz slides using (3-Aminopropyl)methyldichlorosilane (A) and grafted CE using aldol

condensation (B)

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A

B

Figure 5. 2 Spectroscopic results for quartz slides: bare and CE-modifed slides (A) and CE-modifed slide in presence

and absence of Hg ions

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5.2 Nitrite reduction activity of different types of hemoglobin

In our study about electrochemical investigation of nitrite reductase activity (NRA) of hemoglobin,

we proposed a mechanism for the electrochemical reactions occurring at the surface of our

electrode at different potentials. Peak Bred with EP = 834 mV vs Ag/AgCl (at 30 mV/s and pH=7.4)

is assigned to the reduction of nitrite intermediated by hemoglobin. Also, we showed that how

DPV can be employed to investigate NRA of two different type of hemoglobin (Figure 3.6). Here,

we propose that this method can be used to study of many different natural and synthetic

hemoglobins with this relatively fast method. Figure 5.3 shows some preliminary results of cyclic

voltammetry for different hemoglobins.

Figure 5. 3 CV for four different hemoglobins on DDAB modified GC electrodes

-4

-2

0

2

-1.3 -0.5 0.3

I (µ

A)

E (V)

Hum-Hb

Hb A0

Hb A1

Hb S

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5.3 Covalently attached spread bilayer on glassy carbon electrode using diazonium salts

The platform that we proposed for electrochemical investigation of membrane protein by our novel

hybrid bilayer membrane can be improved in different ways for various purposes. One aspect is

improving some properties of the covalently attached molecules (first layer) by choosing the

molecule structure in a way that: the first layer has more movability by choosing longer chains or

different molecular structure, the first layer and/or the second layer molecules consist various polar

and non-polar functional groups to mimic the desired cell membranes, inserting molecules in any

of the first and second layer to prepare a membrane with affinities toward different proteins, and

so many other strategies. One of these strategies may be choosing a phospholipid molecule with

an amine moiety for the first layer. This strategy will result in a covalently attached bilayer with

more similarities to the real cell membranes. One candidate molecule is illustrated in Scheme 5.3

which we believe may give more flexibility to the first layer.

Scheme 5. 3 Chemical structure of 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine which is commercially available

from Sigma-Aldrich (Catalogue #42490 as of January 17th, 2020)

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