1
e-
PROTEINS:
“DOPABLE
SOLID-STATE ELECTRONIC
TRANSPORT MATERIALS
“TRADITIONAL” METHODS TO MEASURE
ELECTRON TRANSFER IN PROTEINS
Electrochemistry
(ML on electrode)
Spectroscopy
(in solution)
Pulseradiolysis
Flashquench
Cyclic
Voltammetry
Chronoamperometry
3
Current (A)
2
1
0
-1
-2
-3
-0.3 -0.2 -0.1 0.0
0.1
0.2
0.3
0.4
Bias vs. SCE (V)
All these measure the Electron transfer rate (s-1)
FROM ELECTRON TRANSFER TO
“SOLID-STATE” ELECTRON TRANSPORT

Goal:


Understand how electron transport (ETp) via proteins
in a 'dry' configuration occurs and what influences it.
Common approaches to measure solid state ETp:
CP-AFM
STM
PROTEIN LAYER
150 nm
400 nm
amide II
CH-stretches
OH strech
3000
2500
2000
1.0
0.8
0.6
0.4
0.2
0.0
400
1500
450
500
-1
550
600
650
700
Wavelength (nm)
Wavenumber (cm )
15
10
5
40
mdeg
IR
amide I
CH-streches
3500
Bacteriorhodopsin (wet)
Bacteriorhodospin (dry ML)
normalized Absorbance (A.U.)
Cytochrome C
0
-5
-10
20
-15
-20
190 200 210 220 230 240 250
mdeg
Normalized Intensity (AU)
PROTEIN LAYER
Wavelength (nm)
0
CD
-20
-40
HSA on quartz
5uM HSA in solution
-60
190
200
210
220
230
Wavelength (nm)
240
250
750
800
850
UV-Vis
Proteins as Electronic Transport Medium?
2
Current Density (A/nm )
Proteins survive partial
Dehydration  Suitable…
1E-8
Proteins are rather efficient
solid-state transport medium
1E-11
1E-14
bR
1E-17
PSI
• Co-factor isscientific
central 
Ultimate
goal:
1E-20
proteins can be doped
Macro ProteinsFerr
Macro Conjugated
Macro Saturated
1E-23
• Amide backbone
maybe
Control
&
predict
involved in elastic transport 1E-26
electron transport
• Functional and dopable  ★★★
across proteins
10
20
30
40
50
60
70
80
90
Molecular Length [Angstrom]
100
PROTEINS ON TODAY’S MENU
Natural electron
transfer proteins
Azurin
Cytochrome C
Not an electron
transfer protein
Serum Albumin
OUR MAIN EXPERIMENTAL APPROACH
Electrical top contact
Idealized
Cartoon!
Conductive substrate
Substrate – Smooth!
 Protein layer – Dense, usually linked by a short linker
 Top electrode – Suitable to contact soft matter

SUBSTRATE



Highly doped Si, Al(ox), Au
Controllable growth of
thin oxide layer on Si
Linker layer (“glue”)
Propyl-silane linker 6-8Å
SiO2 9-10Å
<100> p++-Si (< 0.001 Ω.cm)
TOP ELECTRODE
Au
Hanging Hg drop
Lift off float on (LOFO) - Au
0.2 mm2
~107-109 proteins/contact
CONDUCTIVE PROBE AFM
Less defects
 Force variation

WHAT DOES A NANOSCALE EXPERIMENT
LOOK LIKE?
2 μm
Idealized
Cartoons!
A
10 nm
Metallic substrate
Electrophoretic nanowire assembly of poly-peptide &
protein junctions - suspended nanowire method
protein junction
Azurin
Device figure from G. Noy and Y. Selzer, Angew. Chem. Int. Ed. (2010)
CURRENT-VOLTAGE CHARACTERISTICS OF
THE DIFFERENT PROTEINS
3
2
2
Current density (A/cm )
• Same configuration
Cytochrome c
Azurin
Serum albumin
1
0
• Same temperature
10
1
-1
0.1
-2
SAM propyl-silane
SiO2
Si p++
0.01
1E-3
-3
1E-4
1E-5
-4
1E-6
-1.0
-5
-1.0
-0.5
0.0
Bias (V)
-0.5
0.0
0.5
0.5
1.0
1.0
WHAT IS THE ETP MECHANISM?
Gray and Winkler,
2003
k ET µexp(-bl)
Log conductance
Net decay
k
proteins
ET

1
exp ( 
l
EA
k BT
Isied, JACS 2004
Joachim & Ratner,
PNAS, 2005
1
2
3
4
5
Length (nm)
6
7
8
16
)
WHAT IS THE ETP MECHANISM?

Hopping ………………


Super-exchange ……..


2-step tunneling ???
Thermally activated
Temperatureindependent
Lower βvalues than for
ET in solution
WHAT IS THE ETP MECHANISM?
1. Vary temperature
 2. Modify protein:


Remove the intramolecular cofactor

Replace cofactor
Add
cofactor
Change binding (to electrode)
Change orientation of protein



Not feasible with
ET methodologies
TEMPERATURE DEPENDENCE I-V - AZURIN
Azurin covalently bound to the surface
Sepunaru et al., JACS 2011
CU ION REMOVAL
-12
Holo-Az
ln(J @+0.05V)
-13
-14
-15
300 meV
(
I  Ae
-16
EA
k bT
)
-17
-18
Apo-Az
-19
-20
2
4
6
8
10
12
-1
1000/T [K ]
Sepunaru et al., JACS 2011
CU ION REPLACEMENT
400 300
-12
T [K]
200
100
ln J [+50 mV]
Cu-Az
Ni-Az
-14
Co-Az
-16
Zn-Az
-18
-20
2
4
6
8
10
12
1000/T [K-1]
Unpublished Results
CONNECT TO WHAT WAS DONE BEFORE:
CONDUCTIVE PROBE AFM STUDIES ON AZ
A
All metal
(platinum)
Our setup
Au
Our results (6-15 nN)
Literature results (6 nN)
Au
Tip Bias (V)
J. Davis (Oxford)
setup
Li et al., ACS Nano 2012
TEMPERATURE-DEPENDENT
CONDUCTIVE PROBE AFM
T [K]
T [K]
1.0
380 360
340
320
300
280
260
240
10
4
380
360
340
320
300
Apo-Az
Holo-Az
0.8
280
10
3
10
2
10
1
10
0
I [pA]
0.4
0.2
0.0
2.6
2.8
3.0
3.2
3.4
3.6
3.8
4.0
2.6
2.8
3.2
3.4
3.6
3.8
1000/T [K ]
1000/T [K ]
4
0.6
10
0.4
10
358K
3
0.2
I (pA)
2
0.0
-0.2
10
1
10
268K
0
-0.4
-1.0
3.0
-1
-1
I (nA)
I [nA]
0.6
10
-0.5
0.0
V (V)
0.5
1.0
-1
10
-1.0
-0.5
0.0
0.5
1.0
V (V)
Li et al., ACS Nano 2012
FORCE-DEPENDENT MEASUREMENTS
Holo-Az
Apo-Az
6 nN
9 nN
12 nN
10000
1000
I (pA)
I (pA)
12 nN
9 nN
6 nN
10000
1000
100
10
100
2.8
3.0
3.2
3.4
3.6
3.8
1000/T
4.0
1
2.8
3.0
3.2
3.4
3.6
3.8
4.0
1000/T
Increased force
Increased force
Different ETp mechanism
Increased currents
Same ETp mechanism
Li et al., ACS Nano 2012
TEMPERATURE DEPENDENT I-V WITH CYT C
Cyt C bound electrostatically (physisorbed) to surface
-4
-6
-4
-5
-6
ln(J@0.05V)
ln(J@0.05V)
ln(J@0.05V)
550 meV
-6
-8
-8
100 meV
-7
-10
-8
-10
Iron-free CytC
-12
-9
Holo-CytC
100 meV
100 meV
-14
-10
-12
Apo-CytC
Fe
-11
-16
003.0
10
3.5
10
4.0 20204.5
30
5.0
30
5.5 4040 6.0
1000/T
1000/T
WHAT IS THE ETP MEDIATOR?
Amdursky et al., JACS 2013
CAN WE CONTROL ETP ?
‘DOPING’ SERUM ALBUMIN
-12
Azurin
ln(J@0.05V)
-13
-14
CytC
-15
-16
Apo-Az
-17
HSA
-18
5
10
1000/T
15
20
‘DOPING’ SERUM ALBUMIN WITH HEMIN
Normalized PL
HSA 15uM
HSA-hemin 1-1
HSA-hemin 1-2
HSA-hemin 1-3
280
320
360
400
Wavelength (nm)
440
‘DOPING’ SERUM ALBUMIN WITH HEMIN
0.002
1E-6
2
HSAhemin
1E-5
1E-7
1E-8
-1.0 -0.5 0.0
0.5
Current Density (A/cm )
1E-3
1E-4
2
Current Density (A/cm )
0.004
1.0
0.000
HSA
-0.002
0.004
1E-3
1E-4
1E-5
0.002
1E-6
1E-7
1E-8
0.000
-1.0
-0.5
0.0
0.5
1.0
-0.002
CytC
HSA-hemin
-1.0
-0.5
0.0
Bias (V)
0.5
1.0
-1.0
-0.5
0.0
0.5
1.0
Bias (V)
Amdursky et al., PCCP 2013
‘DOPING’ SERUM ALBUMIN WITH HEMIN
-12
HSA-hemin
CytC electrostatic
-12
-14
ln(J@-0.05V)
95 meV
HSA-hemin
-15
-16
HSA
220 meV
-13
-14
-15
-17
-16
0
5
10
15
20
25
30
0
35
1000/T
0.0008
0.0010
0.0005
kET=18.3 s-1
5
10
kET=4.8
15
20
25
30
35
1000/T
s-1
0.0004
Current
0.0000
Current
ln(J@-0.05V)
-13
0.0000
CytC
-0.0005
HSA-hemin
-0.0004
-0.0008
-0.0010
-0.6
-0.4
-0.2
0.0
Bias
0.2
0.4
0.6
-0.0012
-0.4
-0.2
0.0
0.2
0.4
0.6
Bias
Amdursky et al., PCCP 2013
‘DOPING’ SERUM ALBUMIN WITH HEMIN
WHAT IS THE ETP MEDIATOR?
0.004
2
Current Density (A/cm )
2
Current Density (A/cm )
0.004
0.002
0.000
-1.0
CytC
Iron free CytC
200
Current (nA)
200
Current (nA)
0.000
The conjugated porphyrin
ring,
-0.002
CytC
rather
than the Fe ion
Iron free CytC
-0.004
-0.5 is0.0the0.5main
1.0
-1.0
-0.5
0.0
ETp mediator,
Bias (V)
Bias (V)
while Fe2+/3+ redox controls the
transfer in ET.
-0.002
300
0.002
100
0
-100
HSA-hemin
HSA-PPIX
0.5
1.0
HSA-hemin
HSA-PPIX
100
0
-100
-200
-200
-300
-0.2
-0.1
0.0
0.1
0.2
0.3
Bias vs. SCE (V)
0.4
0.5
-0.2
-0.1
0.0
0.1
0.2
0.3
Bias vs. SCE (V)
0.4
0.5
Amdursky et al., PCCP 2013
Ligand/protein
1/1
2/1
3/1
4/1
0/1
(HSA only)
RA
Absorption (O.D)
0.20
RA/HSA = 4/1
0.15
0.10
RA/HSA = 1/1
0.05
0.00
200
300
400
500
600
Wavelength (nm)
700
RA/HSA = 0
Fluorescent Intensity (A.U.)
0.25
800
Integrated Intensity (A.U.)
‘DOPING’ SERUM ALBUMIN WITH RETINOATE CAN WE INCREASE ETP EFFICIENCY?
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Molar ratio RA/HSA
Ka=4*105
RA/HSA = 4
280
320
360
400
440
480
Wavelength (nm)
Amdursky et al., JACS 2012
‘DOPING’ SERUM ALBUMIN WITH RETINOATE
0/1
1/1
2/1
3/1
4/1
ln(J@-0.05V)
-11
1E-3
-10
2
-10
-9
Current
density (A/cm )
ln(J@-0.05V)
-9
-12
-13
-14
-15
-16
-11
1E-4
-12
1E-5
-13
1E-6
-14
-15
1E-7
0/1220 meV
0/1
1/1140 meV
1/1
2/1125 meV
2/1
3/170 meV
3/1
4/170 meV
4/1
-16
1E-8
-17
-0.6 3.0 -0.43.2
2.8
-0.2
3.4
3.60.0 3.8 0.2
4.0
0.4 4.4 0.6
4.2
Bias(V)-1
1000/T (K )
-17
0
10
20
30
40
50
-1
1000/T (K )
Amdursky et al., JACS 2012
THE POWER OF PROTEIN ‘DOPING’
-10
HSA with 3 retinoates
ln(J@0.05V)
-11
-12
-13
Azurin
-14
-15
-16
HSA
-17
0
5
10
15
20
1000/T
25
30
35
THE IMPORTANCE OF THE CONTACT TO
THE ELECTRODES AND THE ORIENTATION
Electrostatic (physisorbed) vs. Covalent
(chemisorbed) binding
-12.0
-12.5
-13.0
Covalent binding (A15C)
ln (J@-0.05V)

-13.5
CytC
-14.0
-14.5
-15.0
Electrostatic binding (WT)
-15.5
-16.0
0
5
10
15
20
25
30
35
1000/T
Amdursky et al., Submitted
THE IMPORTANCE OF THE CONTACT TO
THE ELECTRODES AND THE ORIENTATION
The importance of the protein’s orientation
-11.5
Azurin (covalent bound)
-12.0
-12.5
ln (J@-0.05V)

Covalent binding (E104C)
-13.0
Covalent binding (A15C)
-13.5
-14.0
-14.5
-15.0
Electrostatic binding (WT)
-15.5
-16.0
0
5
10
15
20
25
30
35
1000/T
Amdursky et al., Submitted
IMPORTANCE OF THE PROTEIN’S ORIENTATION
PREVIOUS STUDIES

Cyt b562
ACS Nano 2012, 355
IMPORTANCE OF THE PROTEIN’S ORIENTATION
5
10
15
20
25
30
35
-12
-14
E104C
-12
G56C
-14
ln(J@-0.05V)
-12
A15C
-14
-12
G23C
-14
-12
V11C
-14
-12
A51C
-14
-12
G37C
-14
-12
Electrostatic
-14
-16
5
10
15
20
1000/T
25
30
35
Amdursky et al., Submitted
Can we use existing models to describe
solid-state type of ETp?
IMPORTANCE OF THE PROTEIN’S ORIENTATION
297K
E104C
2
Curent density @0.05V (A/cm )
5
4
G37C
A51C
G56C
3
2
V11C
A15C
G23C
1
4
E104C
30K
3
2
1
0
26
G37C
V11C
A15C
G56C
A51C
G23C
28
30
32
34
Length (A)
NO distance-current correlation!!!
Amdursky et al., Submitted
IMPORTANCE OF THE PROTEIN’S ORIENTATION
D

Clues from computational modeling
Probably, there is no specific
pathway in the ETp process from
A
one side of the protein to the other
TP – tunneling
pathway
Vs.
APD – atomic
packing density
Amdursky et al., TBP
CONCLUSIONS
Electron transport through proteins can be
measured by solid state configuration, both2 on the
Molecule
Room Temp. R (Ω*nm )
macro and the nano scales for ~4 nm length
 The electron
transport mechanism
Conjugated
~107 - can
109 be
investigated
by
DNA
~107 - 1010



Changing
the temperature
Proteins
~109 - 1011
Modifying
the protein ~1018 - 1022 (extrapolated!)
Saturated
Proteins can be viewed as electronic conducting
material with the possibility of doping
 The contacts to the electrodes and the orientation
of the protein are of prime importance

Thanks to students, PDs & other colleagues
ACKNOWLEDGMENTS
Dr. Ann Erickson
MINERVA FOUNDATION, MUNICH
Abd Elrazek Haj Yahia
Dr. Rotem Har Lavan
Dr. Omer Yaffe
Dr. Ayelet Vilan
Nir K. Kedem
Arava Zohar
Mudi
Sheves
Israel Pecht
43
PORPHYRIN- AND APO-CYTC
40
Normalized Abs.
20
mdeg
Holo-CytC
Porphyrin-CytC
Apo-CytC
Holo-CytC
Porphyrin-CytC
Apo-CytC
0
-20
-40
190
200
210
220
230
Wavelength (nm)
240
250
200
300
400
500
600
Wavelength (nm)
700
800
ET VS. ETP
Waldeck c.s. – PNA length dependence
ACS Nano (2013) 5391