12-Nov-09 1
Ultramicroelectrodes
Electroanalytical Chemistry
Lecture #7
UME?s
? Geometries
? Disk
?Recessed disk
C.S,Henry; I,Fritsch Anal,Chem.
1999,71,550-6.
? Band
? Cylinder
? Materials
? Carbon fiber
UME?s - Advantages
? Reduced capacitance
? Low iR drop
UME?s - Disadvantages
? Noise
? Electrochemical Fouling
? Analyte
?Method of electrode prep?n (epoxy)
? Stray capacitance
? Impurities
? Maintenance
? Cost
? Fragile construction
UME?s - Applications
? Determine [analyte]
? Study reactions
? at low temperatures = frozen
glasses
? in solvents of moderate to high
permittivity w/o electrolyte
? in solvents of high resistance
? in solid state
? in gas phase
UME?s - Applications
(cont?d)
? Biological systems
? Single cells
? organelles
? Fast kinetics
? Development of analytical tools
for electrode/solution interface
? SECM (Bard)
? EQCM
? SERS (Van Duyne)
Scanning Electrochemical
Microscopy (SECM)
? Diffusion is hemispherical
? iss = 4 n F Co* Do a
where a = diam,of UME
? Acquire I vs,tip position (x,y)
? Plot of d vs,tip position
? piezoelectric positioning
? Bipotentiostat
Sample
C W R
Ref.,Bard et al,Science 1991,254,68-74.
d
SECM (cont?d)
? W - 100 nm (diam.)
? Determines resolution
? 2 nm best at present
? Sample
? Conducting
? Insulator
?Mineral
?Biological
? SemiconductorSample
C W R
Common Modes of
Operation
? Feedback
? Positive or negative
? Collection
? flux of species generated/consumed
probed
SECM- Positive
Feedback
? If sample conductive,
we get positive feedback
i > iss
Conductive Sample
C
W
R
R O
SECM - Negative
Feedback
? If sample insulator,
we get negative feedback
i < iss
? Diffusion is hindered
Insulator Sample
C
W
R
R O
OO O
SECM - Operation
? Constant height mode
? tip scanned across substrate,current
monitored
? In situ
? can be done in moist air
SECM - Applications
? Analysis
? Imaging
? Nanolithography
?high precision
?high accuracy
SECM,Reviews
? R.C.Engstrom,C.M.Pharr Anal,
Chem,1989,61,1099A-1104A.
? A.J.Bard,G.Denuault,C.Lee,
D.Mandler,D.O.Wipf Acc,
Chem,Res,1990,23,357-363.
? M.V.Mirkin Anal,Chem,1996,
96,177A-182A.
Electrochemical Quartz Crystal
Microbalances (EQCM)
? Very sensitive mass sensors
? submonolayer
? Design:
? 2 electrodes sandwiching quartz
(piezoelectric)
?oscillating E ? surface of quartz wafer
?oscillation at characteristic frequency
(2 - 20 MHz)
electrode
electrode
Quartz crystal
stuff
EQCM - Principles of
Operation
? Measure change in oscillation
frequency
? Sauerbrey equation:
??f = - 2 (?m/A) n fo2/(?quartz ?quartz)1/2
where
n = overtone number
?quartz = shear modulus (2.95 x 1011
g/cms)
?quartz = density (2.65 g/cm3)
EQCM - Principles of
Operation (cont?d)
? Sauerbrey equation:
??f = - 2 (?m/A) n fo2/(?quartz ?quartz)1/2
? Significance:
? if ?m positive,then ?f ________
? if ?m negative,then ?f ________
? for 300 ?m thick quartz wafer
oscillating at 5 MHz and ?m = 18
ng/cm2 ?f ? 1 Hz (measurable)
EQCM - Principles of
Operation
? Solution exerts a dampening
effect:
?f = - f03/2 [?soln ?soln / (??qaurtz ?quartz)]1/2
where:
?soln = solution viscosity
?soln = solution density
EQCM - Advantages
? Sensitivity
? ng/cm2 Hz
? In situ technique
? not a vacuum method
? Inexpensive
? < $10k
EQCM - Disadvantages
? Selectivity
? mass sensor
?Cannot distinguish what is on surface
EQCM - Applications
? Mechanism of
Electrochemical/Surface
phenomena
? Underpotential deposition (UPD)
?Deposition of first monolayer at E
positive of Eo
? Electroprecipitation
?polymers prep?d electrochemically
different from that prep?d by other
methods
Surface-enhanced Raman
Scattering (SERS)
? 1974 R,Van Duyne
(Northwestern)
? Enhancement 1012 - 1014 comp?d
to nonresonant Raman
SERS - Advantages
? In situ
? Under potential control
? XPS,SEM,etc,not in situ
? Very sensitive
? Good for complex matrices
?Physiological fluids
? Detection limit
? pmole - fmole
?trace analysis
SERS - Advantages
(cont?d)
? Very selective
? Only molecules at/near surface
probed ( up to 16 nm)
? Surface specificity
? Vibrational structural probe
? Info on orientation wrt surface
SERS - Disadvantages
? Reproducibility
? Theoretical understanding of
underlying mechanisms
incomplete
? Limits quantitation
? Limited number of suitable
substrates
? Cu,Ag,Au - best
Raman - History
? 1928 - C.V,Raman,physicist
? C,V,Raman and K,S,Krishnan,
Nature 1928,121,50.,A New
Type of Secondary Radiation”
? 1930 Nobel Prize
http://www.rri.res.in/
Raman - Mechanism
Re
lati
ve
In
ten
sit
y
Frequency
?incident ?incident + ???incident - ??
Stokes Anti-Stokes
E
? = 0 ? = 0
? = 1 ? = 1
virtual virtual
Fewer molecules in
v=1 at RT (kT)
Less intense because
less probable event
h?incident
h(?incident - ??)
Before
After
Raman
? I ?(?ij)2 E
? Tensoral
? Isotropic
? If want to increase I then increase
??ij (molecular)
? E
Resonance Raman (RR)
? Molecular mechanism:
??ij = A/(? - ?o + i?) + B/(? + ?o + i?)
? Resonance
when ? = ?o A term blows up
? Imparts:
?Selectivity
?Sensitivity
Raman - How is it Done?
laser
monochromator
CCD
Camera
lens
Spinning
sample scrambler
grating
SERS - 3 Mechanisms
? Electromagnetic (EM) - Moskovits
? Dominant
? Preferential utility of Cu,Ag,Au
? Image Field - Schatz,van Duyne
? Selection rules
? Adatom - Otto
Electromagnetic
? For noble M with roughness <
?light
? Configuration,d10 s1
? Outer electron behaves as free
electron
?Significance,? has large negative real
component and small imaginary
component
?Ag,?(?) ? -2
Electromagnetic
? Local E for spherical particle of
noble M is:
? Elocal = Eo (?(?) + 2)-1
?so,Elocal is very large
? If elliptical:
? Elocal = Eo (1 + A[?(?) -1])-1
?the more elliptical,the smaller A
?Significance,must go to longer ?
Image Dipole
? Free electrons oscillate with
Eincident and polarize M creating
intense surface Eplasmon inducing a
dipole in the analyte
h?
Parallel,Cancel
h?
Perpendicular,Add
Adatom
? Chemical resonance effect
? Ag + compound = new compound
? Evidence,Ag pyridinium salts
SERS - How Done?
? Enhancement
? Electrochemically
?Oxidation-reduction cycle (ORC)
? oxidize/reduce M in presence of analyte
? Ag/Au colloids
? Potentiostat - if electrochemical
? Raman spectrometer
? Laser
? Monochromator
? CCD detector
EXAMPLE,SERS Detection of
2,4-DNT as Marker for Landmines
? Insanity:
? Installation,removal - 30:1(1995)
? $33B,1100 yrs for removal
? Challenges,
? Time,cost,false positives
? Content,> 99% TNT
? degradation products,2,4-DNT,
1,3-DNB (volatile)
J.M,Sylvia,J.A.Janni,J.D.Klein,K.M.Spencer Anal,Chem,2000,72,5834-40.
http://oneworld.org/gallery/landmines/; photos by Nic Dunlop
EXAMPLE,Cont?d
?Proposal,Raman vapor,sniffer”
? Instrument:
? Diode laser (?exc = 785 nm; 100
mW) with fiber optic probe
? Echelle spectrograph
? CCD camera (air cooled)
? SERS,Au foil (-0.3-1.2 V @
500 mV/s ORC)
? Detection of < 20 ppb 2,4-DNT
in 30 s
SERS Reviews
? R.M.Baum C&E News
1983(Oct.3),22-24.
? T.M.Cotton In Spectroscopy of
Surfaces R.J.H.Clark,R.E.Hester,
Eds.,Wiley,1988,91-153.
? R.L.Garrell Anal,Chem,1989,
61,401A-411A.
? M.J.Weaver,S.Zou,H.Y.H.Chan
Anal,Chem,2000,72,38A-47A.
Bioelectrochemistry
? Relatively new field
?Early ?80?s
? Dates from 1958,Kono and
Nakamura
?Cyt c/Pt
? reduction electrochemically irreversible
Most Work on
? Heme proteins
? Mono-heme
?Cytochrome c (H.A.O,Hill,Hawkridge)
?Myoglobin (Rusling,Taniguchi)
? Several-heme
?Cytochrome c3 (Nikki)
?Hemoglobin (S,Dong)
? Heme peptides
? Microperoxidase-8,-11 (Santucci,Kulys)
? Enzymes (F,Armstrong)
Protein/Electrodes Prepared Using
? Physical immobilization
? Adsorption
? Inclusion
?Sol-gels
? Chemical immobilization
? Cross-linking
?Gluteraldehyde (just like in x-ray)
? Polymeric film
?Polypyrrole (conducting polymer)
?Nafion
Most Work Done Using
? Electrode Materials
? Metals (Au,Pt)
? Carbon
?Glassy (GC)
?Pyrolytic graphite (PG)
? Optically transparent (In-
SnO2/glass) (ITO)
? New Materials
?Boron-doped diamond (BDD)
Approaches to
Bioelectrochemistry
? Direct
? Protein/Electrode (Hawkridge,Rusling)
? Protein/Promoter/Electrode (Hill)
?4,4?-bipyridine
?bis(4-pyridyl) disulfide (BPD)
? Mediated
?Protein/Mediator/Electrode
?Chemically-modified Protein/Electrode (Heller)
? Ferrocene
? Dyes (S,Dong)
Problems for Direct
Electrochemistry
? Irreversible adsorption
? First evidence - SERS (Cotton)
? Denaturation
? Fouling
? Few systems can be
oxidized/reduced
? Sluggish electron transfer rates
Purification is
Important
? 1982 - Cytochrome c (FW
12,384)
? First indication that direct
bioelectrochemistry possible at
naked electrode
Bowden,E.F.; Hawkridge,F.M.; Chlebowski,J.F,
Bancroft,E.E.; Thorp,C.; Blount,H.N,J,Am,
Chem,Soc,1982,104,7641-4.
1CRC,Sanishvili,R.,Volz,K,W.,Westbrook,E,
M.,Margoliash,E,Structure 3 pp,707 (1995)
Promoters
? 1979 first report of
direct electrochemistry
for cyt c/Au modified
with 4,4?-bpy or 1,2-
bis(4-pyridyl)ethylene
? promoter - not directly
involved in electron
transfer
H N
N
S
H
H
H
N
4 - P A T S
Eddowes,M.J.; Hill,H.A.O,J,Am,Chem,Soc,1979,101,4461-2.
Armstrong,F.A.; Hill,H.A.O.; Walton,N.J,Q,Rev,Biophys,1986,3,261-322.
electrode
Cyt c
Purification is Important
? 1993 - Mb (FW 17,000)
in film of
didodecyldimethyl-
ammonium bromide
(DDAB)/Pt,In-SnO2,Au
? 1995 - ultrafiltration used
to remove dimer (FW
34,000)
Rusling,J.F.; Nassar,A.F,J,Am,Chem,
Soc,1993,115,11891-7.
Nassar,A.F.; Willis,W.S.; Rusling,J.F,
Anal,Chem,1995,67,2386-92.
Horse Myoglobin
? Structure:
? FW17,500
? 6-c HS heme; axial ligands,His,
H2O
? Function,oxygen storage and transport
? Fe3+ + e- = Fe2+ -140 mV vs,
Ag/AgCl
? Mb(III)H2O + e- = Mb(II) + H2O
(aquo)met deoxy
6 -c 5-c
? Q,Does H2O dissociate before or
after reductive electron transfer?
1YMB,Evans,S,V.,Brayer,G,D.,High-
resolution study of the three-dimensional
structure of horse heart metmyoglobin,J,
Mol,Biol,213 pp,885 (1990)
Mb Electrochemical Characteristics
S amp l e/E l ect rode E
0
,
V v s,Ag/ AgCl
10
6
x D
0
,
cm
2
/s
10
-4
x k
o
',
cm/ s
Mb(i n DMSO)/ Au -0, 139 + 0.005 0.019 + 0.006 1.7 + 0.5
Mb/ I n
2
O
3
a
-0, 14 0.51 + 0.05 3.0 + 0.5
Mb/ I n
2
O
3
,pH 6,5
b
-0, 140 + 0.005 1.1 + 0.1 8 + 0.5
Mb/ DD AB -PG,
pH 7,5
c
0.05
d
0.37 90 + 3
aTaniguchi,1992
bTaniguchi,1999
cRusling,1993
Spectroelectrochemical
Characterization is Essential
? Classic case,1997 Guadalupe
HRP/GC from DMSO or DMF
? HRP denatures in DMSO and DMF
? Moral,spectroscopy and
electrochemical study must be
accomplished in concert
Guo,Y.; Guadalupe,
A.R,Chem,Commun.
1997,1437-8.
Cofactors
? Nicotinamide adenine
dinucleotide (NADH)
? saw earlier
? Flavin adenine dinucleotide
(FADH)
Me
Me
N
OH
OH
N
OH
N
O
O
N H
O
P
O
OHO
P
O
O
HO
O
OH
N
HO
N
N
NH 2
N
S
S
R
R
S
R
R
Cofactors - NADH
? Nicotinamide adenine dinucleotide (NADH)
? Significance:
?Co-factor for > 300 dehydrogenase enzymes
? Challenges:
?Electrochemical oxidation
?Irreversible adsorption of NAD+
?Electrode deactivation (short term stability)
?High overvoltage (> 1 V)
Abruna,H,see,Anal,Chem,1995,67,3936-44.; Anal,Chem.1996,68,3688-
96.; Anal,Chem,1997,69,4065-75.
Oxidation of NADH at
BDD
? Device characteristics:
? Detection limit 10 nM (n=7)
? Stability - 3 mos.
? Interferents - ascorbic acid
(correctable)
Bioelectrochemistry,The ExacTech
Glucose Electrode
? Amperometric Biosensor,
Mediated electron transfer
electrode
surface
Fc+
Fc
Fc
Glu Ox
Glu Ox
Glu Ox
glucose
glucolactone
+ 2H+
2e-
+0.5 V -0.1 V
e-
Idea,measure current which is correlated with [Glucose]
SBP
? Isolable from soybean seed coat
? soybean U.S,cash crop
? seed coat is waste product
? Single isozyme vs,mixture
(HRP)
? Broad pH stability
? catalytically active from pH 2 - 8
? Thermostable at elevated
temperatures
U.S,Soybean
Crop Value 1974-1999
$ M
illi
on
Statistics,http://www.amsoy.org/; photo,http://mars.cropsoil.uga.edu/csi/
Applications Using SBP
? Medical diagnostics
? Biocatalysis
? manufacture of phenolic resins
(LCD?s,microelectronics,epoxy,
plastics,etc.)
? Hair coloring
? Waste water treatment
? removal of phenol from waste water
and sludge
SBP Sol-Gel H2O2 Sensor
? Need,H2O2 sensor for low pH media
? Food,fermentation
? Approach,
? Enzyme encapsulated (sol-gel)/GC
? Methylene blue - dye mediator
Wang,B.; Li,B.; Wang,Z.; Xu,G.; Wang,Q.; Dong,S,Anal,Chem,1999,
71,1935-9.
SBP Sol-Gel H2O2 Sensor
? Device characteristics:
? Response time - 5 s (fast diffusion)
? Broad pH response (3-7)
? High sensitivity (27.5 ?A/mM)
? Linearity ( 0.02 - 2.6 mM)
? Detection limit (0.5 ?M)
? Reproducibility (1.2% RSD for n=7
at 0.2 mM)
? Interferents
?sulfide,ascorbate,fluoride
Wang,B.; Li,B.; Wang,Z.;
Xu,G.; Wang,Q.; Dong,S,
Anal,Chem,1999,71,
1935-9.
Ultramicroelectrodes
Electroanalytical Chemistry
Lecture #7
UME?s
? Geometries
? Disk
?Recessed disk
C.S,Henry; I,Fritsch Anal,Chem.
1999,71,550-6.
? Band
? Cylinder
? Materials
? Carbon fiber
UME?s - Advantages
? Reduced capacitance
? Low iR drop
UME?s - Disadvantages
? Noise
? Electrochemical Fouling
? Analyte
?Method of electrode prep?n (epoxy)
? Stray capacitance
? Impurities
? Maintenance
? Cost
? Fragile construction
UME?s - Applications
? Determine [analyte]
? Study reactions
? at low temperatures = frozen
glasses
? in solvents of moderate to high
permittivity w/o electrolyte
? in solvents of high resistance
? in solid state
? in gas phase
UME?s - Applications
(cont?d)
? Biological systems
? Single cells
? organelles
? Fast kinetics
? Development of analytical tools
for electrode/solution interface
? SECM (Bard)
? EQCM
? SERS (Van Duyne)
Scanning Electrochemical
Microscopy (SECM)
? Diffusion is hemispherical
? iss = 4 n F Co* Do a
where a = diam,of UME
? Acquire I vs,tip position (x,y)
? Plot of d vs,tip position
? piezoelectric positioning
? Bipotentiostat
Sample
C W R
Ref.,Bard et al,Science 1991,254,68-74.
d
SECM (cont?d)
? W - 100 nm (diam.)
? Determines resolution
? 2 nm best at present
? Sample
? Conducting
? Insulator
?Mineral
?Biological
? SemiconductorSample
C W R
Common Modes of
Operation
? Feedback
? Positive or negative
? Collection
? flux of species generated/consumed
probed
SECM- Positive
Feedback
? If sample conductive,
we get positive feedback
i > iss
Conductive Sample
C
W
R
R O
SECM - Negative
Feedback
? If sample insulator,
we get negative feedback
i < iss
? Diffusion is hindered
Insulator Sample
C
W
R
R O
OO O
SECM - Operation
? Constant height mode
? tip scanned across substrate,current
monitored
? In situ
? can be done in moist air
SECM - Applications
? Analysis
? Imaging
? Nanolithography
?high precision
?high accuracy
SECM,Reviews
? R.C.Engstrom,C.M.Pharr Anal,
Chem,1989,61,1099A-1104A.
? A.J.Bard,G.Denuault,C.Lee,
D.Mandler,D.O.Wipf Acc,
Chem,Res,1990,23,357-363.
? M.V.Mirkin Anal,Chem,1996,
96,177A-182A.
Electrochemical Quartz Crystal
Microbalances (EQCM)
? Very sensitive mass sensors
? submonolayer
? Design:
? 2 electrodes sandwiching quartz
(piezoelectric)
?oscillating E ? surface of quartz wafer
?oscillation at characteristic frequency
(2 - 20 MHz)
electrode
electrode
Quartz crystal
stuff
EQCM - Principles of
Operation
? Measure change in oscillation
frequency
? Sauerbrey equation:
??f = - 2 (?m/A) n fo2/(?quartz ?quartz)1/2
where
n = overtone number
?quartz = shear modulus (2.95 x 1011
g/cms)
?quartz = density (2.65 g/cm3)
EQCM - Principles of
Operation (cont?d)
? Sauerbrey equation:
??f = - 2 (?m/A) n fo2/(?quartz ?quartz)1/2
? Significance:
? if ?m positive,then ?f ________
? if ?m negative,then ?f ________
? for 300 ?m thick quartz wafer
oscillating at 5 MHz and ?m = 18
ng/cm2 ?f ? 1 Hz (measurable)
EQCM - Principles of
Operation
? Solution exerts a dampening
effect:
?f = - f03/2 [?soln ?soln / (??qaurtz ?quartz)]1/2
where:
?soln = solution viscosity
?soln = solution density
EQCM - Advantages
? Sensitivity
? ng/cm2 Hz
? In situ technique
? not a vacuum method
? Inexpensive
? < $10k
EQCM - Disadvantages
? Selectivity
? mass sensor
?Cannot distinguish what is on surface
EQCM - Applications
? Mechanism of
Electrochemical/Surface
phenomena
? Underpotential deposition (UPD)
?Deposition of first monolayer at E
positive of Eo
? Electroprecipitation
?polymers prep?d electrochemically
different from that prep?d by other
methods
Surface-enhanced Raman
Scattering (SERS)
? 1974 R,Van Duyne
(Northwestern)
? Enhancement 1012 - 1014 comp?d
to nonresonant Raman
SERS - Advantages
? In situ
? Under potential control
? XPS,SEM,etc,not in situ
? Very sensitive
? Good for complex matrices
?Physiological fluids
? Detection limit
? pmole - fmole
?trace analysis
SERS - Advantages
(cont?d)
? Very selective
? Only molecules at/near surface
probed ( up to 16 nm)
? Surface specificity
? Vibrational structural probe
? Info on orientation wrt surface
SERS - Disadvantages
? Reproducibility
? Theoretical understanding of
underlying mechanisms
incomplete
? Limits quantitation
? Limited number of suitable
substrates
? Cu,Ag,Au - best
Raman - History
? 1928 - C.V,Raman,physicist
? C,V,Raman and K,S,Krishnan,
Nature 1928,121,50.,A New
Type of Secondary Radiation”
? 1930 Nobel Prize
http://www.rri.res.in/
Raman - Mechanism
Re
lati
ve
In
ten
sit
y
Frequency
?incident ?incident + ???incident - ??
Stokes Anti-Stokes
E
? = 0 ? = 0
? = 1 ? = 1
virtual virtual
Fewer molecules in
v=1 at RT (kT)
Less intense because
less probable event
h?incident
h(?incident - ??)
Before
After
Raman
? I ?(?ij)2 E
? Tensoral
? Isotropic
? If want to increase I then increase
??ij (molecular)
? E
Resonance Raman (RR)
? Molecular mechanism:
??ij = A/(? - ?o + i?) + B/(? + ?o + i?)
? Resonance
when ? = ?o A term blows up
? Imparts:
?Selectivity
?Sensitivity
Raman - How is it Done?
laser
monochromator
CCD
Camera
lens
Spinning
sample scrambler
grating
SERS - 3 Mechanisms
? Electromagnetic (EM) - Moskovits
? Dominant
? Preferential utility of Cu,Ag,Au
? Image Field - Schatz,van Duyne
? Selection rules
? Adatom - Otto
Electromagnetic
? For noble M with roughness <
?light
? Configuration,d10 s1
? Outer electron behaves as free
electron
?Significance,? has large negative real
component and small imaginary
component
?Ag,?(?) ? -2
Electromagnetic
? Local E for spherical particle of
noble M is:
? Elocal = Eo (?(?) + 2)-1
?so,Elocal is very large
? If elliptical:
? Elocal = Eo (1 + A[?(?) -1])-1
?the more elliptical,the smaller A
?Significance,must go to longer ?
Image Dipole
? Free electrons oscillate with
Eincident and polarize M creating
intense surface Eplasmon inducing a
dipole in the analyte
h?
Parallel,Cancel
h?
Perpendicular,Add
Adatom
? Chemical resonance effect
? Ag + compound = new compound
? Evidence,Ag pyridinium salts
SERS - How Done?
? Enhancement
? Electrochemically
?Oxidation-reduction cycle (ORC)
? oxidize/reduce M in presence of analyte
? Ag/Au colloids
? Potentiostat - if electrochemical
? Raman spectrometer
? Laser
? Monochromator
? CCD detector
EXAMPLE,SERS Detection of
2,4-DNT as Marker for Landmines
? Insanity:
? Installation,removal - 30:1(1995)
? $33B,1100 yrs for removal
? Challenges,
? Time,cost,false positives
? Content,> 99% TNT
? degradation products,2,4-DNT,
1,3-DNB (volatile)
J.M,Sylvia,J.A.Janni,J.D.Klein,K.M.Spencer Anal,Chem,2000,72,5834-40.
http://oneworld.org/gallery/landmines/; photos by Nic Dunlop
EXAMPLE,Cont?d
?Proposal,Raman vapor,sniffer”
? Instrument:
? Diode laser (?exc = 785 nm; 100
mW) with fiber optic probe
? Echelle spectrograph
? CCD camera (air cooled)
? SERS,Au foil (-0.3-1.2 V @
500 mV/s ORC)
? Detection of < 20 ppb 2,4-DNT
in 30 s
SERS Reviews
? R.M.Baum C&E News
1983(Oct.3),22-24.
? T.M.Cotton In Spectroscopy of
Surfaces R.J.H.Clark,R.E.Hester,
Eds.,Wiley,1988,91-153.
? R.L.Garrell Anal,Chem,1989,
61,401A-411A.
? M.J.Weaver,S.Zou,H.Y.H.Chan
Anal,Chem,2000,72,38A-47A.
Bioelectrochemistry
? Relatively new field
?Early ?80?s
? Dates from 1958,Kono and
Nakamura
?Cyt c/Pt
? reduction electrochemically irreversible
Most Work on
? Heme proteins
? Mono-heme
?Cytochrome c (H.A.O,Hill,Hawkridge)
?Myoglobin (Rusling,Taniguchi)
? Several-heme
?Cytochrome c3 (Nikki)
?Hemoglobin (S,Dong)
? Heme peptides
? Microperoxidase-8,-11 (Santucci,Kulys)
? Enzymes (F,Armstrong)
Protein/Electrodes Prepared Using
? Physical immobilization
? Adsorption
? Inclusion
?Sol-gels
? Chemical immobilization
? Cross-linking
?Gluteraldehyde (just like in x-ray)
? Polymeric film
?Polypyrrole (conducting polymer)
?Nafion
Most Work Done Using
? Electrode Materials
? Metals (Au,Pt)
? Carbon
?Glassy (GC)
?Pyrolytic graphite (PG)
? Optically transparent (In-
SnO2/glass) (ITO)
? New Materials
?Boron-doped diamond (BDD)
Approaches to
Bioelectrochemistry
? Direct
? Protein/Electrode (Hawkridge,Rusling)
? Protein/Promoter/Electrode (Hill)
?4,4?-bipyridine
?bis(4-pyridyl) disulfide (BPD)
? Mediated
?Protein/Mediator/Electrode
?Chemically-modified Protein/Electrode (Heller)
? Ferrocene
? Dyes (S,Dong)
Problems for Direct
Electrochemistry
? Irreversible adsorption
? First evidence - SERS (Cotton)
? Denaturation
? Fouling
? Few systems can be
oxidized/reduced
? Sluggish electron transfer rates
Purification is
Important
? 1982 - Cytochrome c (FW
12,384)
? First indication that direct
bioelectrochemistry possible at
naked electrode
Bowden,E.F.; Hawkridge,F.M.; Chlebowski,J.F,
Bancroft,E.E.; Thorp,C.; Blount,H.N,J,Am,
Chem,Soc,1982,104,7641-4.
1CRC,Sanishvili,R.,Volz,K,W.,Westbrook,E,
M.,Margoliash,E,Structure 3 pp,707 (1995)
Promoters
? 1979 first report of
direct electrochemistry
for cyt c/Au modified
with 4,4?-bpy or 1,2-
bis(4-pyridyl)ethylene
? promoter - not directly
involved in electron
transfer
H N
N
S
H
H
H
N
4 - P A T S
Eddowes,M.J.; Hill,H.A.O,J,Am,Chem,Soc,1979,101,4461-2.
Armstrong,F.A.; Hill,H.A.O.; Walton,N.J,Q,Rev,Biophys,1986,3,261-322.
electrode
Cyt c
Purification is Important
? 1993 - Mb (FW 17,000)
in film of
didodecyldimethyl-
ammonium bromide
(DDAB)/Pt,In-SnO2,Au
? 1995 - ultrafiltration used
to remove dimer (FW
34,000)
Rusling,J.F.; Nassar,A.F,J,Am,Chem,
Soc,1993,115,11891-7.
Nassar,A.F.; Willis,W.S.; Rusling,J.F,
Anal,Chem,1995,67,2386-92.
Horse Myoglobin
? Structure:
? FW17,500
? 6-c HS heme; axial ligands,His,
H2O
? Function,oxygen storage and transport
? Fe3+ + e- = Fe2+ -140 mV vs,
Ag/AgCl
? Mb(III)H2O + e- = Mb(II) + H2O
(aquo)met deoxy
6 -c 5-c
? Q,Does H2O dissociate before or
after reductive electron transfer?
1YMB,Evans,S,V.,Brayer,G,D.,High-
resolution study of the three-dimensional
structure of horse heart metmyoglobin,J,
Mol,Biol,213 pp,885 (1990)
Mb Electrochemical Characteristics
S amp l e/E l ect rode E
0
,
V v s,Ag/ AgCl
10
6
x D
0
,
cm
2
/s
10
-4
x k
o
',
cm/ s
Mb(i n DMSO)/ Au -0, 139 + 0.005 0.019 + 0.006 1.7 + 0.5
Mb/ I n
2
O
3
a
-0, 14 0.51 + 0.05 3.0 + 0.5
Mb/ I n
2
O
3
,pH 6,5
b
-0, 140 + 0.005 1.1 + 0.1 8 + 0.5
Mb/ DD AB -PG,
pH 7,5
c
0.05
d
0.37 90 + 3
aTaniguchi,1992
bTaniguchi,1999
cRusling,1993
Spectroelectrochemical
Characterization is Essential
? Classic case,1997 Guadalupe
HRP/GC from DMSO or DMF
? HRP denatures in DMSO and DMF
? Moral,spectroscopy and
electrochemical study must be
accomplished in concert
Guo,Y.; Guadalupe,
A.R,Chem,Commun.
1997,1437-8.
Cofactors
? Nicotinamide adenine
dinucleotide (NADH)
? saw earlier
? Flavin adenine dinucleotide
(FADH)
Me
Me
N
OH
OH
N
OH
N
O
O
N H
O
P
O
OHO
P
O
O
HO
O
OH
N
HO
N
N
NH 2
N
S
S
R
R
S
R
R
Cofactors - NADH
? Nicotinamide adenine dinucleotide (NADH)
? Significance:
?Co-factor for > 300 dehydrogenase enzymes
? Challenges:
?Electrochemical oxidation
?Irreversible adsorption of NAD+
?Electrode deactivation (short term stability)
?High overvoltage (> 1 V)
Abruna,H,see,Anal,Chem,1995,67,3936-44.; Anal,Chem.1996,68,3688-
96.; Anal,Chem,1997,69,4065-75.
Oxidation of NADH at
BDD
? Device characteristics:
? Detection limit 10 nM (n=7)
? Stability - 3 mos.
? Interferents - ascorbic acid
(correctable)
Bioelectrochemistry,The ExacTech
Glucose Electrode
? Amperometric Biosensor,
Mediated electron transfer
electrode
surface
Fc+
Fc
Fc
Glu Ox
Glu Ox
Glu Ox
glucose
glucolactone
+ 2H+
2e-
+0.5 V -0.1 V
e-
Idea,measure current which is correlated with [Glucose]
SBP
? Isolable from soybean seed coat
? soybean U.S,cash crop
? seed coat is waste product
? Single isozyme vs,mixture
(HRP)
? Broad pH stability
? catalytically active from pH 2 - 8
? Thermostable at elevated
temperatures
U.S,Soybean
Crop Value 1974-1999
$ M
illi
on
Statistics,http://www.amsoy.org/; photo,http://mars.cropsoil.uga.edu/csi/
Applications Using SBP
? Medical diagnostics
? Biocatalysis
? manufacture of phenolic resins
(LCD?s,microelectronics,epoxy,
plastics,etc.)
? Hair coloring
? Waste water treatment
? removal of phenol from waste water
and sludge
SBP Sol-Gel H2O2 Sensor
? Need,H2O2 sensor for low pH media
? Food,fermentation
? Approach,
? Enzyme encapsulated (sol-gel)/GC
? Methylene blue - dye mediator
Wang,B.; Li,B.; Wang,Z.; Xu,G.; Wang,Q.; Dong,S,Anal,Chem,1999,
71,1935-9.
SBP Sol-Gel H2O2 Sensor
? Device characteristics:
? Response time - 5 s (fast diffusion)
? Broad pH response (3-7)
? High sensitivity (27.5 ?A/mM)
? Linearity ( 0.02 - 2.6 mM)
? Detection limit (0.5 ?M)
? Reproducibility (1.2% RSD for n=7
at 0.2 mM)
? Interferents
?sulfide,ascorbate,fluoride
Wang,B.; Li,B.; Wang,Z.;
Xu,G.; Wang,Q.; Dong,S,
Anal,Chem,1999,71,
1935-9.
