T U Berlin Nov. 14, 2014
Towards Protein-based Bio-Electronics
Electron Transfer &
Solid-State Electronic Transport
Fundamental differences and similarities
with
Mordechai Sheves , Israel Pecht
Nadav Amdursky, Lior Sepunaru
Debora Marchak, Noga Friedman +++++
Support
Minerva Foundation, Munich
Israel Min. of Science
“SOLID-STATE” ELECTRON TRANSPORT (ETP)
Some proteins “survive”
partial dehydration
A
Idealized cartoon
metal
linker layer
substrate / contact
Macro-electrode options for soft matter:
Hg, “ready-made” metallic pad, evaporated Pb
Hg drop
pad
Lift-off float-on (LOFO) e.g., Au, PEDOT-PSS
0.2 mm2
106 - 1010 proteins/contact
Replacing or removing the Cu ion 
Zn-azurin or apo-Azurin
Az
Apo-Az
Zn-Az
1.0
0.5
Current vs. voltage
I-V [ln (I)-V]
@ RT
0.0
-0.5
1E-5
-1.0
1E-6
1E-7
-1.5
Current (A)
Current (A)
Example of ETp measurement: Azurin
-2.0
-2.5
1E-8
1E-9
Az
Apo-Az
Zn-Az
1E-10
1E-11
A
Idealized cartoon
metal
1E-12
-3.0
-1.0
-3.5
-1.0
-0.5
0.0
-0.5
0.0
0.5
1.0
Bias Voltage (on metal) [V]
0.5
Bias Voltage (on metal) [V]
linker layer
substrate / contact
1.0
Ron et al, JACS 2010
Temperature dependent conduction
of “any (> ~2 nm) peptide skeleton”
200
100
-12
2
Current density [mA/cm ]
400 300
T [K]
-13
-14
-15
BSA
apo-Azurin
-16
-17
-18
2
4
6
8
10
12
-1
1000/T [K ]
5
Electron Transport Mechanism
Temperature
independent
Thermally
activated
Bovine
Serum
Albumin
1E-8
2
Current Density (A/nm )
“SOLID-STATE” ELECTRON TRANSPORT (ETP)
Some proteins “survive”
partial dehydration
m
A
500 µm
Metallic Pad
Hg Drop
1E-17
1E-10
1E-12
1E-14
33
34
35
1E-20
1E-22
1E-24
~33 Å
(Azurin)
~6-9 Å
Saturated
Conjugated
Proteins
Amdursky et al.,
Adv. Mater. 2014
10
metal
linker layer
32
1E-18
1E-26
substrate / contact
1E-18
1E-16
Idealized cartoon
50 nm
300-450 µm
Mercapto-propyl linker
macroscopic
20
30
40
50
60
Length (Å)
70
80
90 100
We (can) also use nanoscale contacts; let’s
take a closer look at such experiments:
2 μm
Idealized
Cartoons!
A
10 nm
Metallic substrate
Nanoscale contacts – Azurin
Apo-Az
Holo-Az
Current (nA)
1
holo-Az 12nN
apo-Az 12nN
holo-Az 6nN
0.1
0.01
apo-Az 6nN
1E-3
1E-4
1E-5
-0.5
0.0
0.5
Bias (V)
Davis group, JMC 2005
WIS group, ACS Nano 2012
9
Resistance (G)
Applied force-dependent conductance
25
Resistance (G)
20
15
@RT
25
WT-bR
WT-Azurin
20
15
Azurin
10
5
bR
0
10
0
5
10
15
Applied Force (nN)
5
Plastic regime
0
0
5
10
15
Applied force (nN)
20
25
Li et al. ACS Nano,
10 2012
Mukhopadhyay et al., ACS Nano (2014)
How does Electron Transport (ETp)
differ from Electron Transfer (ET)?
ETp
e-
ET
hν
ee-
Spectroscopy
In solid state
Electrochemistry
In solution
11
How does Electron Transport (ETp)
differ from Electron Transfer (ET)?
ETp is measured with electronically conducting electrodes that
• are ionically blocking
• have delocalized electron systems (affects reorganization energy)
ET is measured without electrodes (or with one ionically conducting,
electronically blocking contact)
How does Electron Transport (ETp)
differ from Electron Transfer (ET)?
ETp is measured with electronically conducting electrodes that
• are ionically blocking
• have delocalized electron systems (affects reorganization energy)
ET is measured without electrodes (or with one ionically conducting,
electronically blocking contact)
-----------------------------------------------------------------------
ETp is measured on proteins outside their natural environment
• in partially “dry” state, with only tightly bound water kept
(but with natural conformation closely preserved)
ET is measured with protein in, or partially exposed to solution.
How does Electron Transport (ETp)
differ from Electron Transfer (ET)?
ETp is measured with electronically conducting electrodes that
• are ionically blocking
• have delocalized electron systems (affects reorganization energy)
ET is measured without electrodes (or with one ionically conducting,
electronically blocking contact)
-----------------------------------------------------------------------
ETp is measured on proteins outside their natural environment
• in partially “dry” state, with only tightly bound water kept
(but with natural conformation closely preserved)
ET is measured with protein in, or partially exposed to solution.
-----------------------------------------------------------------------
ETp: no redox reaction required  can study ETp close to equilibrium (@0.05 V)
ET : redox reaction required (coupled to ion transport for charge balance)
How does Electron Transport (ETp)
differ from Electron Transfer (ET)?
ETp is measured with electronically conducting electrodes that
• are ionically blocking
• have delocalized electron systems (affects reorganization energy)
ET is measured without electrodes (or with one ionically conducting,
electronically blocking contact)
-----------------------------------------------------------------------
ETp is measured on proteins outside their natural environment
• in partially “dry” state, with only tightly bound water kept
(but with natural conformation closely preserved)
ET is measured with protein in, or partially exposed to solution.
-----------------------------------------------------------------------
ETp: no redox reaction required  can study ETp close to equilibrium (@0.05 V)
ET : redox reaction required (coupled to ion transport for charge balance)
------------------------------------------------------------------------
BUT ETp may be differ from ET if
• pressure is applied (e.g., in SPM)
• significant  (> 1-1.5 V bias voltage) is imposed
• electronic current flows
• …………………………
………
How does Electron Transport (ETp)
differ from Electron Transfer (ET)?
• Effect of protein redox activity (for CytC)
• Redox site effect on conduction (for Az)
(also can add redox site in bR)
• Effect of protein-electrode coupling
Normalized Fluorescence (a.u)
Case Study – I
‘Doping’ serum albumin with hemin
& comparison with Cyt C
HSA
HSA-hemin
280
320
360
400
440
Wavelength (nm)
Some ETp-ET differences :
• Effect of protein redox activity
‘Doping’ serum albumin with hemin
& comparison with Cyt C
1E-6
2
HSAhemin
1E-5
0.002
Current Density (A/cm )
1E-3
1E-4
2
Current Density (A/cm )
0.004
1E-7
1E-8
-1.0 -0.5 0.0
0.5
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
0.5
1.0
Bias (V)
-1.0
-0.5
0.0
0.5
1.0
Bias (V)
Some ETp-ET differences :
• Effect of protein redox activity
Amdursky et al., PCCP 2013
‘Doping’ serum albumin with hemin
& comparison with Cyt C
-12
HSA-hemin
CytC electrostatic
-12
95 meV
ln(J@-0.05V)
ln(J@-0.05V)
-13
-14
HSA-hemin
-15
-16
HSA
220 meV
-13
-14
-15
-17
-16
10
5
0
30
25
20
15
1000/T
0.0008
0.0010
0.0005
kET ≈ 5
15
10
5
0
35
20
1000/T
s-1
0.0004
kET ≈ 18 s-1
Current
Current
0.0000
0.0000
CytC
-0.0005
-0.0004
HSA-hemin
-0.0008
-0.0010
-0.6
Amdursky et al., PCCP 2013
-0.4
-0.2
0.0
Bias
0.2
0.4
0.6
-0.0012
-0.4
-0.2
0.0
0.2
Bias
0.4
0.6
25
30
35
‘Doping’ serum albumin with hemin
& comparison with Cyt C
Cyt C electrostatically
bound (physisorbed) to surface
-4
-6
ln(J@0.05V)
ln(J@0.05V)
-6
-8
-8
100 meV
Iron-free CytC
-10
-10
-12
Holo-CytC
-14
-12
Apo-CytC
Fe
-16
00
10
10
2020
3030
4040
1000/T
1000/T
Some ETp-ET differences :
• Effect of protein redox activity
Amdursky et al., JACS 2013
‘Doping’ serum albumin with hemin
& comparison with Cyt C
0.004
2
Current Density (A/cm )
2
Current Density (A/cm )
0.004
0.002
0.000
-0.002
0.000
The conjugated porphyrin ring, not
-0.002
the Fe ion, is the main ETp mediator,
-0.004
while in ET the Fe2+/3+ redox
-1.0process
-0.5
0.0
Bias (V)
controls
the electron transfer. Bias (V)
CytC
Iron free CytC
-1.0
0.002
-0.5
0.0
0.5
1.0
CytC
Iron free CytC
300
200
HSA-hemin
HSA-PPIX
0.5
1.0
HSA-hemin
HSA-PPIX
Current (nA)
Current (nA)
200
100
0
-100
100
0
-100
-200
-200
-300
-0.2
-0.1
Amdursky et al., PCCP 2013
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
CASE STUDY-II
AZURIN
Some ETp – ET differences:
• Redox site effect on conduction
Cu ion removal
-12
Holo-Az
ln(J @+0.05V)
-13
-14
-15
EA )
I  Ae kbT
(
300 meV
-16
-17
-18
Apo-Az
-19
-20
2
4
6
8
10
-1
1000/T [K ]
Sepunaru et al., JACS 2011
12
AZURIN
Some ETp – ET differences:
• Redox site effect on conduction
400 300
-12
T [K]
200
100
ln J [+50 mV]
Cu-Az
Ni-Az
-14
Co-Az
-16
Zn-Az
-18
Cu ion replacement
-20
2
4
6
8
1000/T [K-1]
TBP
10
12
AZURIN
Some ETp – ET differences:
• Redox site effect on conduction
holo-Az - Protonated
-12
ln(J@0.05V)
-13
-14
holo-Az - Deuterated
-15
-16
apo-Az - Deuterated
-17
apo-Az - Protonated
-18
0
5
10
15
20
1000/T
Amdursky et al., PNAS 2013 and TBP
25
30
35
3
AZURIN
+2
Cu
+1
Cu
2
+0.5V
+0.2V
+0.05V
-0.05V
-0.2V
-0.5V
-7
-8
1
0
-9
ln(J)
-1
-2
-3
-4
@RT
-5
-6
-1.0
-0.5
0.0
0.5
Cu(I) vs. Cu(II) Az
1.0
Bias (V)
-10
-11
-12
2
-7
+0.5V
+0.2V
+0.05V
-0.05V
-0.2V
-0.5V
-8
-9
ln(J)
4
6
8
10
12
14
16
1000/T
-10
-11
-12
-13
5
10
15
1000/T
TBP
20
25
Some ETp – ET differences:
• Redox site effect on conduction
CASE STUDY-III
ETP WITH CYT C MUTANTS
Some ETp – ET differences:
• Effect of transport distance ?
with Dmitry Dolgikhd & Rita Chertkovad
Shemyakin-Ovchinnikov Inst. Bioorg. Chem., RAS
Carlo Bortolotti, U Modena
Amdursky et al., PNAS 2014
ETP WITH CYT C MUTANTS
-11.5
-12.0
ln (J@-0.05V)
-12.5
Covalent binding (E104C)
-13.0
Covalent binding (A15C)
-13.5
-14.0
Covalent binding (A51C)
-14.5
-15.0
Electrostatic binding (WT)
-15.5
-16.0
Amdursky et al., PNAS 2014
0
5
10
15
Some ETp – ET differences:
• Effect of transport distance ?
20
1000/T
25
30
35
cytochrome C
Azurin (covalent bound)
5
E104C
297K
2
4
G37C
A51C
G56C
3
A15C
G23C
1
4
E104C
30K
3
2
1
0
26
G37C
V11C
A15C
G56C
A51C
G23C
28
30
Length (A)
electrode
32
34
2
2
V11C
Curent density @0.05V (A/cm )
Curent density @0.05V (A/cm )
ETP WITH CYT C MUTANTS
4.0
E104C
3.5
3.0
2.5
2.0
G37C
G56C
V11C
1.5
A15C
1.0
A51C
0.5
G23C
0.0
4
5
6
7
8
9
10
11
12
Heme edge-electrode closest proximity (A)
Cys
contact
to electrode
Amdursky et al., PNAS 2014
Some ETp – ET differences:
• effect of partial transport distance
ETP WITH CYT C MUTANTS
-11.5
-12.0
ln (J@-0.05V)
-12.5
Covalent binding (E104C)
-13.0
Covalent binding (A15C)
-13.5
-14.0
Covalent binding (A51C)
-14.5
-15.0
Electrostatic binding (WT)
-15.5
-16.0
Amdursky et al., PNAS 2014
0
5
10
Some ETp – ET differences:
• Protein-electrode coupling
15
20
1000/T
25
30
35
cytochrome C
Azurin (covalent bound)
ETP WITH CYT C MUTANTS
Some ETp – ET differences:
• Protein-electrode coupling
Amdursky et al., PNAS 2014
Let’s try to put things in perspective:
Protein vs. conjugated & saturated molecule conduction
Saturated CP-AFM
Saturated STM
Saturated Electromigration
Conjugated CP-AFM
Conjugated STM
Proteins CP-AFM
Proteins STM
2
Current Density (A/nm )
1E-5
nm-scale
1E-6
1E-7
1E-8
1E-9
@RT
1E-10
1E-11
1E-12
1E-13
1E-14
1E-15
0
10
20
30
40
Length (Å)
50
60
70
Amdursky et al., Progress Report
Adv. Mater. 9-2014
Resistance (G)
but … remember applied force-dependent conductance
25
Resistance (G)
20
15
25
WT-bR
WT-Azurin
20
15
10
5
0
10
0
5
10
15
Applied Force (nN)
5
Plastic regime
0
0
5
10
15
Applied force (nN)
20
25
W. Li et al. ACS Nano, 2012
32
S. Mukhopadhyay et al., ACS Nano (2014)
Let’s try to put things in perspective:
2
Current Density (A/nm )
Protein vs. conjugated & saturated molecule conduction
Saturated CP-AFM
Saturated STM
Saturated Electromigration
Conjugated CP-AFM
Conjugated STM
Proteins CP-AFM
Proteins STM
2
Current Density (A/nm )
1E-5
nm-scale
1E-6
1E-7
1E-8
1E-9
1E-10
1E-11
1E-12
1E-13
@RT
1E-14
1E-15
0
10
20
30
40
Length (Å)
50
60
70
1E-8
macroscopic
1E-10
1E-17
1E-12
1E-14
1E-16
1E-18
32
33
34
35
1E-18
1E-20
1E-22
1E-24
Saturated
Conjugated
Proteins
@RT
1E-26
10
20
30
40
50
60
70
80
90 100
Length (Å)
Magnitudes of ETp via proteins more like those via
conjugated than those via saturated Amdursky
molecules!!
et al., Progress Report
Adv. Mater. 9-2014
Measured/equivalent J (A/nm2)
Let’s try to put things in perspective:
Compare ET to ETp results on proteins
Spectroscopy
Electrochemistry
CP-AFM
STM
Macroscopic
1E-10
1E-12
1E-14
kET = J . constant
1E-16
@RT
1E-18
1E-20
1E-22
20
40
60
80
100
Length (Å)
ETp allows measuring (also) over long distances;
Amdursky et al.,
Report
ET explores shorter distances Adv.Progress
Mater. 9-2014
Let’s try to put things in perspective:
Compare ET to ETp results on proteins
1E12
J = kET / constant
1E10
STM
-1
kET (s )
1E8
1000000
10000
Macroscopic
100
1
@RT
0.01
20
40
60
Length (Å)
80
100
Amdursky et al.,
Progress Report
Adv. Mater. 9-2014
So, from where do we start re. ET vs. ETp ?
Proteins are good conduction media; WHY / HOW ?
• Need for redox-active proteins
ETp does not require redox activity; never?
• Redox site effect on ETp
Can be minimal (check with ETp-vibrational spectr.)
when is cofactor important (e.g., Cu(I) effect)?
So, from where do we start re. ET vs. ETp ?
Proteins are good conduction media; WHY / HOW ?
• Need for redox-active proteins
ETp does not require redox activity; never?
• Redox site effect on ETp
Can be minimal (check with ETp-vibrational spectr.)
when is cofactor important (e.g., Cu(I) effect)?
• Importance of transport distance in ETp ??
So, from where do we start re. ET vs. ETp ?
Proteins are good conduction media; WHY / HOW ?
• Need for redox-active proteins
ETp does not require redox activity; never?
• Redox site effect on ETp
Can be minimal (check with ETp-vibrational spectr.)
when is cofactor important (e.g., Cu(I) effect)?
• Importance of transport distance in ETp ??
• Importance of contact – cofactor distance & coupling in ETp:
~barrier height; “main-lining”  temp.-independent conduction ?
use/ make proteins with cofactor close to likely contact area;
can we identify coupling spectroscopically: IETS, vibr. spectr.-ETp?
So, what did we learn till now?
Proteins do not behave as electronic insulators; WHY / HOW ?
(Several) proteins can be investigated in the solid state,
while essentially remaining intact.
Both temperature-independent and thermally activated
mechanisms contribute to conduction.
Redox/prosthetic groups are important for conduction, but ….
different from ET, ETp - conduction doesn’t require
redox activity (~~large vs. small polaron hopping)
Re. peptides…., you can ask us
39
Thanks to
Mudi
Sheves
Some of the people
Israel Pecht
Sidney
Cohen
40
Noga
Friedman
40
0.6
0.4
0.2
0.0
400
450
500
550
600
650
700
750
800
normalized Absorbance (A.U.)
0.8
850
1.0
0.8
0.6
0.4
bR Photocycle
0.2
0.0
Photochemical
300
400
Wavelength (nm)
bR solution - light on
bR solution - light off
bR dry monolayer - light on
bR dry monolayer - light off
0.4
0.2
0.0
-0.2
-0.4
-0.6
300
400
500
600
700
800
normalized emission intensity (AU)
normalizedAbsorption
Absorbance (A.U.)
UV-Vis.
1.0
Az solution
dry Az film
500
600
700
Thermal(nm)
Wavelength
800
bR568
1.0
Az solution
Az film
0.8
0.6
0.4
M412
0.2
0.0
300
320
340
360
Fluorescence
Spectrum
Difference
normalized Absorbance
(A.U.)
Bacteriorhodopsin (wet)
Bacteriorhodospin (dry ML)
UV-Vis. Absorption
Can the proteins “survive”
(partial) dehydration ?
380
Wavelength (nm)
Wavelength (nm)
Bacteriorhodopsin (bR)
Azurin
Ron et al, JACS 2010
Toolbox
Room temperature, ambient conditions
Monolayer characterization
Linker layer
Conductive substrate
- AFM;
TEM  Cryo-ED;
- Ellipsometry; UV-Visible Abs.;
- FT-IR;
Surface Potential
XRR
Fluorescence
Preparation of
“Solid-State” Protein Junctions
• Substrate - smooth !
Metal or Semiconductor
• Linker layer - self-assembled
short molecule monolayer with
functional terminal group
• Protein layer – should be
dense; orientation ??
Linker layer
Conductive substrate
idealized
cartoon
=NH2, Br, SH
Preparation of
“Solid-State” Protein Junctions
• Substrate - smooth !
Metal or Semiconductor
Electrical top contact
• Linker layer - self-assembled
short molecule monolayer with
functional terminal group
• Protein layer – should be
dense; orientation ??
Linker layer
Conductive substrate
idealized
cartoon
• Top contact –
deposition
and composition compatible
with ‘soft’ biological material
Electrical Transport Characteristics: I-V
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
-2.5
8
bR
Az
6
Mean Current (A)
4
2
0
-2
-4
-6
-8
-10
-12
-14
-1.0
-0.5
0.0
0.5
1.0
-1.0
Voltage [V]
-0.5
0.0
0.5
1.0
Voltage [V]
Mean Current (nA)
Mean Current (A)
(Mean and SD of 30 junctions)
100
80
60
40
20
0
-20
-40
-60
-80
-100
-120
-140
BSA
-1.0
-0.5
0.0
Voltage [V]
0.5
1.0
Ron et al, JACS 2010
Monolayer characterization
12 nm
bR
50 nm
bR
5 nm
A
z
50 nm
A
z
5 nm
BSA
50 nm
BSA
500 nm x 500 nm scans, AFM height images (AC mode)
Ellipsometry: SiOx: 11-12 Å, organo-silane linkers: 6-7 Å
C o v e r a g e
>
90 %
bR
Protein height: 5 nm
Az
Protein height: 3.6 nm
BSA
Protein height: 4 nm
AFM Height:
AFM height: ~ 3.5 nm
AFM height: ~ 4 nm
5 nm
rms roughness:0.35-0.4 nm rms roughness:0.55-0.6 nm
Ron et al, JACS 2010
Recap of experimental approach:
probe the proteins sandwiched between two conducting electrodes 
Metal/Bridge/Metal configuration.
1) MACROSCOPIC CONTACTS
Keep reproducibility in mind:
• Highly doped Si slides
• Controllable growth of
thin oxide layer
Propyl-silane linker 6-8Å
SiO2 9-10Å
150 nm
• Linker layer (‘glue”)
<100> p++ (<0.001 Ohm cm)
400 nm
but …
intimate 5 µm2 contact to a 0.5 nm2 /monolayer of molecules ?
Sure, just contact each grass leaf (~3 cm2) on 70×100 m2 soccer field
[Akkerman]
still, higher over-all currents  large measuring ability gain
…..
Is also a Cartoon!!
…..
contact
…..
…..
…..
…..
Electron transfer and “Solid-state” Conduction
of proteins
Alkyl, peptide,
DNA….
conduction
protein
What is the ETp mechanism?
• Hopping
• Thermally activated
• Super-exchange
• Temperature independent
• “2-step tunneling” • Low beta values
Electron transfer and conduction relations
Case I: off resonance tunneling
Conductance
Franck-Condon density of
states and electron transfer
rate
Width of
molecular
energy levels
Usually broadened
due to interactions
with the leads
Conductance
via off
resonance
tunneling is
temperature
independent.
Charge transfer upon contact
between the systems is not taken
into account!
A. Nitzan – J Phys chem A 2001
T [K]
400 300
200
100
-4
Bacteriorhodopsin
Current density [mA/cm2]
ln J [+50 mV Bias]
-6
-8
Pre-melting transition
-10
-12
Temperature
independent
-14
-16
Conformational
change
-18
-20
2
4
6
8
10
-1
1000/T [K ]
12
60
Tuning electron transport in Bacteriorhodopsin
400 300
100
2
Current density [mA/cm ]
-8
200
T [K]
-10
-12
-14
-16
-18
-20
2
4
6
8
-1
1000/T [K ]
10
12
61
Creating 2 pathways
400 300
100
2
Current
Current density
[mA/cm ]
density [mA/cm
-8
200
T [K]
-10
-12
Reconstitution
-14
-16
-16
-18
-18
-20
-20
22
44
66
88
-1
-1
1000/T
[K
1000/T [K ]]
10
10
12
12