electrochemical nanopatterning and microsystems
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Applied Biotechnology Innovation Centre
Electrochemical Nanopatterning and
Microsystems
Ioanis Katakis
Department of Chemical Engineering, ATIC Technology Innovation Centre, Universitat Rovira i Virgili, Tarragona, Spain
November 29, 2010
NANOJASP 2010, Barcelona
Applied Biotechnology Innovation Centre
DEFINING TERMS
HOLDING EVERYTHING TOGETHER
THE TRANSDUCER
THE BIOMOLECULE(S)THE TRANSDUCTION
CHEMISTRY
MODULATING ACTIVITIES
CONTROLLING RATES OF REACTION AND MASS TRANSPORT
• The generic bioelectronic element
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MODULE 1
--
MODULE 2
++ + MODULE 3
-- -
MODULE 4
Os
Os Os+ +
MODULE 5
MODULE 6...
-
-+
+
+
Os
Os
Os
+
+-
-
-
• Each module can be any of the components. You aim at flexibility and control of outcome.
MODULAR IMMOBILISATION...
• Narváez et al. Biosens Bioelectr 15:43-52 (2000)
• Narváez et al. J Electroanal Chem 430:227-33 (1997)
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• Rational manipulation of bioregeneration kinetics
Au / MPS / RP / PSS / FDH
+
+Os
Os
Os
S SO3-
S SO3-
SO3-
SO3-
SO3-
ORDERED IMMOBILISATION: Multilayer self-assembled redox polyelectrolyte-FDH architecture in gold electrodes
average catalytic current = 1.04 µA RSD = 16 % n = 6
0 0.1 0.2 0.3 0.4 0.5
E / V vs Ag/AgCl
0
0.4
0.8
1.2
I / µ
AAu / MPS / RP / PSS / (B / PSS)n / FDH
S SO3-
S SO3-
+
+Os
Os
Os
SO3-
SO3-
SO3-
SO3-
SO3-
SO3-+
+
+
+
Fructose modular electrodes
…TO CONTROL PROPERTIES
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SUPRAMOLECULAR ARCHITECTURES FOR COMPLEX BIOSENSING, AMPLIFICATION, AND
CATALYSIS TASKS
• Popescu et al. J Electroanal Chem 464:208-14 (1999)
Lipid
Os-Phendion-surfactant
GLDH
NAD+
Cholesterol
Substrate
Product
Dehydrogenase
NAD(P)+
NAD(P)H
Mediatorred
Mediatorox
Electrode e-
APPLY JUDICIOUSLY MOLECULAR ENGINEERING
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• CONTACT AND CONTACT-LESS SPOTTING WELL DEVELOPED FOR 100+ mm RESOLUTION
• BIOMOLECULE PHOTOLITHOGRAPHY FOR HIGH DENSITY APPLICATIONS
BUT WHAT ABOUT PATTERNING?
WHAT IF WE COULD USE ELECTROCHEMICALLY-DRIVEN METHODS FOR BOTH PATTERNING AND DETECTION?
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• Colloidal gold: a versatile nano/module
- - - --
---
-++
+
Electrostatic forces
Adsorption phenomena
sDative binding
“NANO” ENGINEERING SPATIAL INTELLIGENCE
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• Campàs & Katakis Int J Env Anal (2004), PCT/EP2003/000262 (2002)
• Campàs & Katakis Sens. & Actuators B (2006)
gold colloidoligonucleotide
colloidal gold-oligonucleotideselective deposition
deposition
conjugation
NANOCOLLOID SYNTHESIS AND MODIFICATION FOR NANOPATTERNING AND ARTIFICIAL
INTELLIGENCE
PATTERNING BIOLOGICAL PROPERTIES
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Electrochemistry on SPE
e-
0
5
10
15
20
I (nA
)
1.2V (compl)
no E (compl)
1.2V (4-mut)
no E (4-mut)
ARE THEY FUNCTIONAL?
• Concept works but high exists non-specific adsorption
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GOx
GOx
GOx
GOx
GOx
• Adding properties (transduction) to nanopatterns
MOLECULAR ENGINEERING OF “NANO”...
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AuAu
PEG
SH
PEG
SH
PEG
SH
PEG
SH
Thioctic acid SAM
e1 (-1.2V)
desorption of
Thioctic acid
e1 (+0.8V)
selective adsorption
of HRP-Os-Au
e2 -1.2V)
selective
desorption
The deprotection of the second electrode avoids the non-specific adsorption detected.
e2 (+1.2V)
deposition of
Gox-Os-Au
There is only a 3,4% of non-specific
response from the second electrode
H2O
2
e-
Amperometric
detection of HRP (e1) Non-specific adsorption was detected
on the second electrode
Glucos
e
e-
Amperometric detection
of GOx (e2) 5% of non-specific response was
detected from the first electrode
• Amperometric detection
of electrodeposited
biomolecules
…AND IMPROVING SELECTIVITY
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SS
HO
O
OO
O
SS
HO
O
OO
O
SS
HO
O
OO
O
SS
HO
O
OO
O
SS
O
OO
O
O
O
Au
OO
O
O
O
H
SS SS
HO
O
OO
O
SS
HO
O
OO
O
SS
HO
O
OO
O
SS
HO
O
OO
O
Au
+700mV
ONE STEP FURTHER: PATTERNS AT MOLECULAR LEVEL
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0.477 0.577 0.677 0.777 0.877 0.977 1.077-6-0.069x10
-6-0.019x10
-60.031x10
-60.081x10
-60.131x10
-60.181x10
-60.231x10
-60.281x10
-60.331x10
-60.381x10
-60.431x10
E / V
i / A
Peak around +0.6V
Electrochemical deprotection was nearly complete within one scan
DOES IT WORK?
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0
100
200
300
400
500
600
Compoundimmobilisation
Gox(non-specif ic) Gox(AfterElectroactivation)
Fre
qu
en
cy c
han
ge(H
z)
• EQCM data shows hope (but still 30% non specificity)
IS IT SELECTIVE?
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• Resist coating• First laser exposure• First biomolecule coating • Second laser exposure• Second biomolecule coating• Polyelectrolyte blocking
Laser ablation or lithography work equally well
AND YET ANOTHER METHOD OF PATTERNING
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• Layer optimisation: GOX(first layer) SAOX (second layer)
Activity of glucose for every step of PE layer deposition
0
0,00000005
0,0000001
0,00000015
0,0000002
0,00000025
0,0000003
0,00000035
0,0000004
0,00000045
0 5 10 15 20 25 30
glucose
i/A
native
1st layer
2nd layer
3rd layer
GOXGOX 2h.
POS+GOX
SOX 2h.
POS+SOX
• After third layer of polyelectrolyte the response for enzyme decrease 30%.
BIOPHOTOLITHGRAPHY: CATALYSIS(1)
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GOX (Max. Response 856A)
SOX (Max. Response 10 nA)
• Layer optimisation: GOX(first layer) SAOX (second layer)
0
0.0000001
0.0000002
0.0000003
0.0000004
0.0000005
0.0000006
0.0000007
0.0000008
0.0000009
0 2 4 6 8 10 12 14
substrate(mM)
i/A
Gox and SAOx response with Glucose
GOX
SAOx
GOXGOX 2h.
POS+GOX
SOX 2h.
POS+SOX
BIOPHOTOLITHGRAPHY: CATALYSIS(1)
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GOX (Max. Response 0A) No crosstalk.
SOX (Max. Response 101 nA)
• Layer optimisation: SAOx (first layer) GOX (second layer)
0.00E+00
1.00E-08
2.00E-08
3.00E-08
4.00E-08
5.00E-08
6.00E-08
7.00E-08
8.00E-08
9.00E-08
1.00E-07
0 5 10 15 20 25 30 35 40substrate(mM)
uA
Gox and SAOx response with Sarcosine
GOx
SAOx
SOX
GOX2h.
POS+GOX
SOX 2h.
POS+SOX
BIOPHOTOLITHGRAPHY: CATALYSIS(2)
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1st electrode
(Sample)
2nd
electrode
(Control)
AuAu
Deprotection of e1 with
UV
Incubation of e1 with
redox polymer and
streptavidine
Immobilisaion of
biotinylated mutated
capture probe in e2
Deprotection of e2 with UVIncubation of e2 with
redox polymer and
streptavidine
Immobilisation of
biotinylated wild capture
probe in e1
Incubation of both
electrode with the
biotinylated target
Incubation of both
electrode with
streptavidine-HRP
Amperometric detection of
both electrodes
HRP HRP
H2O2 e-
BSA blocking in
e1BSA blocking in
e2
IDE: Higher signal was obtained from the electrode with wild probe (1.7A), 0.4A was obtained from the mutated probe and 0.3 A from the control without target.
Also in CE higher signal was obtained
from the electrode with wild probe
(50nA), while 0nA was obtained from
electrode where mutated probe was
immobilised
BIOPHOTOLITHGRAPHY: HYBRIDISATION
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• HCG amperometric detection through sandwich assay
1st electrode
(Sample) 2nd electrode
(Control)
AuAu
HRP
H2O2 e-
e1
deprotection
Incubation with redox
polymer and anti-HCG in
e1
BSA blocking in
e1
Incubation of HCG target
and biotinylated anti-HCG
in e1
e2 deprotectionIncubation with redox
polymer and anti-HCG in
e2
BSA blocking in
e2
Incubation of biotinylated
anti-HCG in e2 (control)
Incubation of both
electrodes with
streptavidine-HRP
Amperometric detection of HRP
IDE: lower signal was obtained comparing with DNA, however as in DNA wafers the control was lower (2nA) than the sample (40nA)
Also in CE there is a lower
response from the control,
nevertherless the signal is lower.
BIOPHOTOLITHGRAPHY: MOLECULAR RECOGNITION(1)
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• T4 amperometric detection through competition assay
1st electrode
(Control)
2nd electrode
(Sample)
AuAu
H2O2 e-
e1
deprotection
Incubation with redox
polymer and BSA-T4 in e1BSA blocking in
e1
Incubation with anti-T4 in e1
(Control)e2 deprotection
Incubation with redox
polymer and BSA-T4 in e2BSA blocking in
e2
Incubation with T4 and anti-T4
in e2
Incubation of both
electrodes with anti-
Rabbit Igg-HRP
Amperometric detection of HRP
HRP
A competition assay was carried
out to detect T4. 94.2nA was
obtained from the control and
47.3nA from the sample
BIOPHOTOLITHGRAPHY: MOLECULAR RECOGNITION(2)
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SHSH SH
SH
HRP
e-
H2O2
SH
SHSH
SH
A difference of 1,5µA between sampleand blank and a limit of detection of 6.31fmoles was obtained
MODULATING ACTIVITY: RECOGNITION TO SENSING
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H2O2
e-
HRP
H2O2
e-
- 57% and 23% of signal displaced in colourimetric and electrochemical displacement- Fast response: 2 minutes in electrochemical displacement
AND FROM SENSING TO FACILE SENSING
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Os+Os+
Os+Os+
Os+Os+
Os+Os+
Os+Os+
Os+Os+
- - --- -
+ + +++ +- - --- - }N1
+ + +++ +
+ + +++ + }N2
- - --- -
Glucose oxidase produces H2O2
Peroxidase uses H2O2 and consumes electrons at E2
Glucose oxidase produces electrons at E1
e-
e-
TOWARDS THE ULTIMATE NANOMACHINE(?): SELF POWERED, SELF PROPELLED, SELF PROPAGATING
INTEGRATING TECHNOLOGIES FOR MORE FUNCTIONS
• Pescador et al Langmuir (2008)
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Using same principles for versatile microsystem operation
ELECTRODEPOSITION AS PART OF OPERATION
A
B
A
B
• Mata at al Electroch. Acta (2009)
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THE TEAM
• Panagiotis Argitis
NCSR DEMOKRITOS
• Mònica Campàs
• Mònica Mir
• Srujan Dondapati
• Pablo Lozano
Universitat Rovira i Vrigili
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Our work is financed by:
• MICROPROTEIN (Patterning and arraying)
• HEALTHY AIMS (Fuel cells)
• CELSITIVE (Pathogen Detection)
• CIDEM and our Clients
• URV
THE MONEY
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THANK YOU FOR YOUR
ATTENTION
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