Towards Protein-based Bio-Electronics Electron Transfer & Solid - State Electronic Transport Fundamental differences and similarities with Mordechai Sheves,

Towards Protein-based Bio-Electronics

Electron Transfer &

Solid-State Electronic Transport

Fundamental differences and similaritieswith

Mordechai Sheves , Israel PechtNadav Amdursky, Lior Sepunaru

Debora Marchak, Noga Friedman +++++

T U Berlin Nov. 14, 2014

Support

Minerva Foundation, MunichIsrael Min. of Science

Some proteins “survive”partial dehydration

“SOLID-STATE” ELECTRON TRANSPORT (ETP)

substrate / contactlinker layer

Ametal

Idealized cartoon

pad

Lift-off float-on (LOFO) e.g., Au, PEDOT-PSS

Macro-electrode options for soft matter: Hg, “ready-made” metallic pad, evaporated Pb

Hg drop

HgSubstrate

Backcontact

Si

Pb

~1nm

0.2 mm2 106 - 1010 proteins/contact

-1.0 -0.5 0.0 0.5 1.0-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

Cur

rent

(A

)

Bias Voltage (on metal) [V]

Az

-1.0 -0.5 0.0 0.5 1.0

1E-12

1E-11

1E-10

1E-9

1E-8

1E-7

1E-6

1E-5

Cur

rent

(A

)


Az

-1.0 -0.5 0.0 0.5 1.0-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

Cur

rent

(A

)


Apo-Az

-1.0 -0.5 0.0 0.5 1.0

1E-12

1E-11

1E-10

1E-9

1E-8

1E-7

1E-6

1E-5

Cur

rent

(A

)


Apo-Az

-1.0 -0.5 0.0 0.5 1.0-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

Cur

rent

(A

)


Zn-Az

-1.0 -0.5 0.0 0.5 1.0

1E-12

1E-11

1E-10

1E-9

1E-8

1E-7

1E-6

1E-5

Cur

rent

(A

)


Zn-Az

Ron et al, JACS 2010

Replacing or removing the Cu ion Zn-azurin or apo-Azurin

Example of ETp measurement: Azurin

Current vs. voltageI-V [ln (I)-V]

@ RT


Ametal

Idealized cartoon

Temperature dependent conduction of “any (> ~2 nm) peptide skeleton”

2 4 6 8 10 12-18

-17

-16

-15

-14

-13

-12

BSA apo-Azurin

Cu

rren

t d

ensi

ty [

mA

/cm

2 ]

1000/T [K-1]

400 300 200 100T [K]

5

Temperature independent

Thermally activated

Electron Transport Mechanism

BovineSerum

Albumin

Some proteins “survive”partial dehydration

“SOLID-STATE” ELECTRON TRANSPORT (ETP)

10 20 30 40 50 60 70 80 90 1001E-26

1E-24

1E-22

1E-20

1E-18

1E-16

1E-14

1E-12

1E-10

1E-8 macroscopic

Saturated Conjugated Proteins

Cur

rent

Den

sity

(A/n

m2 )

Length (Å)

32 33 34 351E-18

1E-17


Ametal

Idealized cartoon

Amdursky et al., Adv. Mater. 2014

IdealizedCartoons!

We (can) also use nanoscale contacts; let’s take a closer look at such experiments:

A

10 nm

Metallic substrate

2 μm

9

Nanoscale contacts – Azurin

Holo-Az Apo-Az

-0.5 0.0 0.51E-5

1E-4

1E-3

0.01

0.1

1 holo-Az 12nN

holo-Az 6nNapo-Az 12nN

Cur

rent

(nA

)

Bias (V)

apo-Az 6nN

WIS group, ACS Nano 2012Davis group, JMC 2005

10

0 5 10 15 20 250

5

10

15

20

25R

esis

tan

ce (

G

)

Applied force (nN)

0 5 10 150

5

10

15

20

25 WT-bR WT-Azurin

Res

ista

nce

(G

)

Applied Force (nN)

Applied force-dependent conductance

Elastic regime

Plastic regime

Li et al. ACS Nano, 2012Mukhopadhyay et al., ACS Nano (2014)

Azurin

bR

@RT

11

e-

e-

hν

In solid state In solution

ETp ET

Spectroscopy Electrochemistry

e-

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.






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)






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• ………………………… ………


• 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


280 320 360 400 440

Nor

mal

ized

Flu

ores

cenc

e (a

.u)

Wavelength (nm)

HSA

HSA-hemin

Case Study – I‘Doping’ serum albumin with hemin

& comparison with Cyt C

Some ETp-ET differences :

• Effect of protein redox activity

-1.0 -0.5 0.0 0.5 1.0

1E-8

1E-7

1E-6

1E-5

1E-4

1E-3

-1.0 -0.5 0.0 0.5 1.0

-0.002

0.000

0.002

0.004

Cur

rent

Den

sity

(A

/cm

2 )

Bias (V)

HSA

HSA-hemin

-1.0 -0.5 0.0 0.5 1.0

-0.002

0.000

0.002

0.004

Cur

rent

Den

sity

(A

/cm

2 )

Bias (V)

CytC HSA-hemin

-1.0 -0.5 0.0 0.5 1.01E-8

1E-7

1E-6

1E-5

1E-4

1E-3

‘Doping’ serum albumin with hemin& comparison with Cyt C

Amdursky et al., PCCP 2013



0 5 10 15 20 25 30 35

-17

-16

-15

-14

-13

-12

ln

(J@

-0.0

5V)

1000/T

HSA

HSA-hemin

0 5 10 15 20 25 30 35-16

-15

-14

-13

-12

HSA-hemin CytC electrostatic

ln(J

@-0

.05V

)1000/T

95 meV

220 meV

-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6

-0.0010

-0.0005

0.0000

0.0005

0.0010

Cur

rent

Bias

-0.4 -0.2 0.0 0.2 0.4 0.6-0.0012

-0.0008

-0.0004

0.0000

0.0004

0.0008

Cur

rent

Bias

HSA-heminCytC

kET ≈ 18 s-1

kET ≈ 5 s-1



0 10 20 30 40

-12

-10

-8

-6

ln

(J@

0.05

V)

1000/T

100 meV

Fe

0 10 20 30 40-16

-14

-12

-10

-8

-6

-4

ln

(J@

0.05

V)

1000/T

Iron-free CytC

Holo-CytC

Apo-CytC

Cyt C electrostatically bound (physisorbed) to surface

Amdursky et al., JACS 2013




-1.0 -0.5 0.0 0.5 1.0

-0.002

0.000

0.002

0.004

CytC Iron free CytC

Bias (V)

Cur

rent

Den

sity

(A

/cm

2 )

-1.0 -0.5 0.0 0.5 1.0-0.004

-0.002

0.000

0.002

0.004

HSA-hemin HSA-PPIX

Cur

rent

Den

sity

(A

/cm

2 )

Bias (V)

-0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5

-200

-100

0

100

200 HSA-hemin HSA-PPIX

Cur

rent

(nA

)

Bias vs. SCE (V)

-0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5

-300

-200

-100

0

100

200

300 CytC Iron free CytC

Cur

rent

(nA

)

Bias vs. SCE (V)

The conjugated porphyrin ring, not the Fe ion, is the main ETp mediator,

while in ET the Fe2+/3+ redox process controls the electron transfer.



)(A

TkE

AeI b

2 4 6 8 10 12

-20

-19

-18

-17

-16

-15

-14

-13

-12

Holo-Az

1000/T [K-1]

ln(J

@+

0.05

V)

Cu ion removal

300 meV

Sepunaru et al., JACS 2011

2 4 6 8 10 12

-20

-19

-18

-17

-16

-15

-14

-13

-12

Apo-Az

Holo-Az

1000/T [K-1]

ln(J

@+

0.05

V)

CASE STUDY-IIAZURINSome ETp – ET differences:

• Redox site effect on conduction

2 4 6 8 10 12-20

-18

-16

-14

-12

ln J

[+

50 m

V]

1000/T [K-1]

400 300 200 100T [K]

Cu-Az

Ni-Az

Co-Az

Zn-Az

TBP

Cu ion replacement

Some ETp – ET differences: • Redox site effect on conduction

AZURIN

0 5 10 15 20 25 30 35

-18

-17

-16

-15

-14

-13

-12holo-Az - Protonated

holo-Az - Deuterated

apo-Az - Deuterated

1000/T

ln(J

@0.

05V

)

apo-Az - Protonated

Amdursky et al., PNAS 2013 and TBP

AZURINSome ETp – ET differences: • Redox site effect on conduction

-1.0 -0.5 0.0 0.5 1.0

-6

-5

-4

-3

-2

-1

0

1

2

3

Cur

rent

(A

)

Bias (V)

Cu+2

Cu+1

2 4 6 8 10 12 14 16

-12

-11

-10

-9

-8

-7

+0.5V +0.2V +0.05V -0.05V -0.2V -0.5V

ln(J

)

1000/T

5 10 15 20 25

-13

-12

-11

-10

-9

-8

-7

+0.5V +0.2V +0.05V -0.05V -0.2V -0.5V

ln(J

)

1000/T

Cu(I) vs. Cu(II) Az

Some ETp – ET differences:

• Redox site effect on conduction TBP

@RT

AZURIN

CASE STUDY-IIIETP WITH CYT C MUTANTS

Amdursky et al., PNAS 2014

with Dmitry Dolgikhd & Rita ChertkovadShemyakin-Ovchinnikov Inst. Bioorg. Chem., RASCarlo Bortolotti, U Modena


• Effect of transport distance ?

0 5 10 15 20 25 30 35

-16.0

-15.5

-15.0

-14.5

-14.0

-13.5

-13.0

-12.5

-12.0

-11.5

Covalent binding (A51C)

Azurin (covalent bound)

Covalent binding (E104C)


Electrostatic binding (WT)

ln

(J@

-0.0

5V)

1000/T

cyto

chro

me

C


ETP WITH CYT C MUTANTS


• Effect of transport distance ?

26 28 30 32 340

1

2

3

41

2

3

4

5

A51C

G23C

G56CA15C

G37CV11C

E104C

A51C

G23C

G56C

A15C

G37C

V11C

E104C

Cu

ren

t de

nsi

ty @

0.0

5V

(A

/cm

2 )

Length (A)

30K

297K

4 5 6 7 8 9 10 11 120.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

A51C

G23C

G56CA15C

G37CV11C

E104C

Cur

ent d

ensi

ty @

0.05

V (A

/cm

2 )

Heme edge-electrode closest proximity (A)

electrode

Cys contact

to electrodeAmdursky et al., PNAS 2014


• effect of partial transport distance


0 5 10 15 20 25 30 35

-16.0

-15.5

-15.0

-14.5

-14.0

-13.5

-13.0

-12.5

-12.0

-11.5


Azurin (covalent bound)

Covalent binding (E104C)


Electrostatic binding (WT)

ln

(J@

-0.0

5V)

1000/T

cyto

chro

me

C



• Protein-electrode coupling




• Protein-electrode coupling


0 10 20 30 40 50 60 701E-15

1E-14

1E-13

1E-12

1E-11

1E-10

1E-9

1E-8

1E-7

1E-6

1E-5

nm-scale

Saturated CP-AFM Saturated STM Saturated Electromigration Conjugated CP-AFM Conjugated STM Proteins CP-AFM Proteins STM

Cur

rent

Den

sity

(A

/nm

2 )

Length (Å) Amdursky et al., Progress ReportAdv. Mater. 9-2014

Let’s try to put things in perspective: Protein vs. conjugated & saturated molecule conduction

@RT

32

0 5 10 15 20 250

5

10

15

20

25R

esis

tan

ce (

G

)

Applied force (nN)

0 5 10 150

5

10

15

20

25 WT-bR WT-Azurin

Res

ista

nce

(G

)

Applied Force (nN)

but … remember applied force-dependent conductance

Elastic regime

Plastic regime

W. Li et al. ACS Nano, 2012S. Mukhopadhyay et al., ACS Nano (2014)

0 10 20 30 40 50 60 701E-15

1E-14

1E-13

1E-12

1E-11

1E-10

1E-9

1E-8

1E-7

1E-6

1E-5

nm-scale

Saturated CP-AFM Saturated STM Saturated Electromigration Conjugated CP-AFM Conjugated STM Proteins CP-AFM Proteins STM

Cur

rent

Den

sity

(A

/nm

2 )

Length (Å)

Magnitudes of ETp via proteins more like those via conjugated than those via saturated molecules!!

Let’s try to put things in perspective: Protein vs. conjugated & saturated molecule conduction

Amdursky et al., Progress ReportAdv. Mater. 9-2014

10 20 30 40 50 60 70 80 90 1001E-26

1E-24

1E-22

1E-20

1E-18

1E-16

1E-14

1E-12

1E-10

1E-8 macroscopic

Saturated Conjugated Proteins

Cur

rent

Den

sity

(A/n

m2 )

Length (Å)

32 33 34 351E-18

1E-17

@RT

@RT

ETp allows measuring (also) over long distances; ET explores shorter distances

20 40 60 80 1001E-22

1E-20

1E-18

1E-16

1E-14

1E-12

1E-10

Length (Å)

Spectroscopy Electrochemistry CP-AFM STM Macroscopic

Mea

sure

d/eq

uiva

lent

J (

A/n

m2)

Let’s try to put things in perspective: Compare ET to ETp results on proteins

kET = J . constant

Amdursky et al.,Progress Report

Adv. Mater. 9-2014

@RT

20 40 60 80 1000.01

1

100

10000

1000000

1E8

1E10

1E12

Length (Å)

k ET (

s-1)

STM

CP-AFM

Macroscopic

SpectroscopicElectrochem.

Let’s try to put things in perspective: Compare ET to ETp results on proteins

J = kET / constant

Amdursky et al.,Progress Report

Adv. Mater. 9-2014

@RT

• Need for redox-active proteinsETp does not require redox activity; never?

• Redox site effect on ETpCan 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 ?



• Importance of transport distance in ETp ??




• 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

40

40

Some of the people Israel Pecht

MudiSheves

Thanks to

Sidney Cohen

Noga Friedman

Can the proteins “survive” (partial) dehydration ?

bR568

M412

Photochemical

Thermal

bR Photocycle

AzurinBacteriorhodopsin (bR)

UV-V

is. Ab

sorp

tion

Fluore

scen

ce

400 450 500 550 600 650 700 750 800 850

0.0

0.2

0.4

0.6

0.8

1.0n

orm

aliz

ed A

bso

rban

ce (

A.U

.)

Wavelength (nm)

Bacteriorhodopsin (wet) Bacteriorhodospin (dry ML)

300 400 500 600 700 800

0.0

0.2

0.4

0.6

0.8

1.0

nor

mal

ized

Ab

sorb

ance

(A

.U.)

Wavelength (nm)

Az solution

300 400 500 600 700 800

0.0

0.2

0.4

0.6

0.8

1.0

Az solution dry Az film

300 320 340 360 380

0.0

0.2

0.4

0.6

0.8

1.0

norm

aliz

ed e

mis

sion

inte

nsit

y (A

U)

Wavelength (nm)

Az solution

300 320 340 360 380

0.0

0.2

0.4

0.6

0.8

1.0 Az solution Az film


UV-V

is.

Ab

sorp

tion

bR dry monolayer - light on bR dry monolayer - light off

bR solution - light off

300 400 500 600 700 800-0.6

-0.4

-0.2

0.0

0.2

0.4

nor

mal

ized

A

bso

rban

ce (

A.U

.)

Wavelength (nm)

bR solution - light on

Diff

ere

nce

Sp

ect

rum

Toolbox

Room temperature, ambient conditions

Monolayer characterization

Conductive substrate

Linker layer

- AFM; TEM Cryo-ED; XRR

- Ellipsometry; UV-Visible Abs.; Fluorescence

- FT-IR; Surface Potential

Preparation of “Solid-State” Protein Junctions


Linker layer

• Substrate - smooth ! Metal or Semiconductor

• Linker layer - self-assembled short molecule monolayer with functional terminal group

• Protein layer – should be dense; orientation ??

idealizedcartoon

Si

OO

O

X=NH2, Br, SH

Preparation of “Solid-State” Protein Junctions


Linker layer

Electrical top contact

• Substrate - smooth ! Metal or Semiconductor

• Linker layer - self-assembled short molecule monolayer with functional terminal group

• Protein layer – should be dense; orientation ??

• Top contact – deposition and composition compatible with ‘soft’ biological material

idealizedcartoon

Electrical Transport Characteristics: I-V(Mean and SD of 30 junctions)

-1.0 -0.5 0.0 0.5 1.0

-140-120-100-80-60-40-20

020406080

100

Mea

n C

urre

nt (

nA)

Voltage [V]

-1.0 -0.5 0.0 0.5 1.0

-2.5-2.0-1.5-1.0-0.50.00.51.01.52.02.53.03.5

Mea

n C

urre

nt (A

)

Voltage [V]

-1.0 -0.5 0.0 0.5 1.0-14

-12

-10

-8

-6

-4

-2

0

2

4

6

8

Mea

n C

urre

nt (A

)

Voltage [V]

bR Az

BSA


bRProtein height: 5 nm

AFM Height: 5 nm

Ellipsometry: SiOx: 11-12 Å, organo-silane linkers: 6-7 Å

Monolayer characterization

AzProtein height: 3.6 nm

AFM height: ~ 3.5 nm

rms roughness:0.35-0.4 nm

BSAProtein height: 4 nm

AFM height: ~ 4 nm

rms roughness:0.55-0.6 nm

bR Az BSA

bR Az BSA

bR Az BSA

bR Az BSA

500 nm x 500 nm scans, AFM height images (AC mode)

12 nm 5 nm 5 nm

50 nm 50 nm 50 nm

C o v e r a g e > 90 %


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

• Linker layer (‘glue”) <100 >p++ (<0.001 Ohm cm)

SiO2 9-10Å

Propyl-silane linker 6-8Å

400 nm

150 nm

…..

…..

….. …..

…..

contact

…..

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]

but …

Is also a Cartoon!!

still, higher over-all currents large measuring ability gain

Electron transfer and “Solid-state” Conduction of proteins

Alkyl, peptide, DNA….

protein

conduction

What is the ETp mechanism?

• Hopping• Super-exchange• “2-step tunneling”

• Thermally activated• Temperature independent• Low beta values

ConductanceWidth of

molecular energy levels

Franck-Condon density of states and electron transfer

rate

Conductance via off resonance tunneling is temperature independent.

Electron transfer and conduction relationsCase I: off resonance tunneling

Usually broadened due to interactions

with the leads A. Nitzan – J Phys chem A 2001

Charge transfer upon contact between the systems is not taken

into account!

2 4 6 8 10 12

-20

-18

-16

-14

-12

-10

-8

-6

-4ln

J [

+50

mV

Bia

s]

1000/T [K-1]

400 300 200 100T [K]

Linear regime

Conformational change

Pre-melting transition

Temperature independent

Bacteriorhodopsin

Curr

ent d

ensi

ty [m

A/cm

2 ]

60

2 4 6 8 10 12

-20

-18

-16

-14

-12

-10

-8

Cu

rren

t d

ensi

ty [

mA

/cm

2 ]

1000/T [K-1]

400 300 200 100T [K]

EA=160 meV

WT bR

Apo bR

EA=500 m

eV2 4 6 8 10 12

-20

-18

-16

-14

-12

-10

-8

Cu

rren

t d

ensi

ty [

mA

/cm

2 ]

1000/T [K-1]

400 300 200 100T [K]

Tuning electron transport in Bacteriorhodopsin

61

2 4 6 8 10 12

-20

-18

-16

-14

-12

-10

-8

Cu

rre

nt

de

ns

ity

[mA

/cm

2 ]

1000/T [K-1]

400 300 200 100T [K]

2 4 6 8 10 12

-20

-18

-16

-14

-12

-10

-8

Cu

rren

t d

ensi

ty [

mA

/cm

2 ]

1000/T [K-1]

400 300 200 100T [K]

Reconstitution

Creating 2 pathways

Towards Protein-based Bio-Electronics Electron Transfer & Solid - State Electronic Transport Fundamental differences and similarities with Mordechai Sheves,

Documents

electron transfer et

delocalized electron

electrodes thatare

oneionically conducting

reorganization energy

ron et

acs nano

azurin br