microelectrochemistry for chemical messenger detectiondopamine cyclic voltammogram t i. advantages:...
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Microelectrochemistry for Chemical Messenger Detection
Haynes Group Tutorial11/15/2007
Tutorial Outline
• Introduction to microelectrodes• Fast-scan cyclic voltammetry• Constant potential amperometry• Bioanalytical applications of
microelectrochemistry
Relevant Redox Reactions
NH2
OH
OH
NH2
O
O-2e-, -2H+
Carbon-Fiber Microelectrodes
Carbon Fiber
Glass
Carbon Fiber
Glass
Disk Cylinder
SA ~ 50 μm2 SA ~ 1000 μm2
Carbon fibers made from eitherpitch precursor or polyacrylonitrile.
+
Microelectrode Fabrication
Microelectrode Fabrication
Cure
Polish
AmperometryCarbon Fiber
Glass
Carbon Fiber
Glass
i
t
StimulatingPipette
Microelectrode
+650 mVvs. Ag/AgCl
Diffusion at Disk Microelectrodes
Diffusion occurs in two dimensions*radially wrt the axis of symmetry*normal to the plane of the electrode
So, current density is not uniform across theface of the disk (greater at the edge).
Current-time relationship has 3 regimes:1. Short time scale (diffusion layer << r0): diffusion has a
semi-infinite linear character2. Intermediate time scale (diffusion layer ~ r0): radial
diffusion becomes important and the current is larger than for a continuation of pure linear diffusion
3. Long time scales (diffusion layer >> r0): the current approaches a steady state
r0
Diffusion-Limited Current
12
1 12 2
*
( ) O Od
nFAD Ci ttπ
=
n = number of electrons exchangedF = Faraday’s constant (96,485 C/mole)A = electrode area (cm2)DO = diffusion coefficient for species O (cm2/s)C*
O = initial concentration of the reducible analyte O (M)
**
00
4 4O Oss O O
nFAD Ci nFD C rrπ
= =
The Cottrell equation describes change in current with respect to time at a planarelectrode in a controlled potential experiment under diffusion control.
Modified Cottrell equation for disk microelectrode:
TT
R
R R
LL--DOPADOPA
TyrTyr
Single synapseSingle synapse
DADA
DADA
Intact tissueIntact tissue
T
T
T
T
TT
T
T
T
TT
T
T
T T
T
T
T
T
T
T
RR
R
R
R
R
R
R
R
R
R
R
R
RR
R
R
R
R
R
5 μm
Dopamine Synthesis, Release, and Uptake
DADA10 µm
Glass Seal
Carbon Fiber
10 µm
Glass Seal
Carbon Fiber
DopamineDopamine--oo--quinonequinone
--2 e2 e--
+2 e+2 e--
DopamineDopamineNH2
OHOH
NH2
OO
Diffusion at Cylinder Microelectrodes
Diffusion occurs in one dimension
Current-time relationship has 2 regimes:1. Short time scale (diffusion layer << electrode curvature):
diffusion follows Cottrell equation2. Long time scales:
*
20 0
2 4 where ln
O O Oqss
nFAD C D tir r
ττ
= =
Modifying Carbon-Fiber Microelectrodes
• Combating fouling of the carbon surface– Apply CV in 0.1 M NaOH
• Treatments for increased sensitivity/selectivity– overoxidation– nafion– polypyrrole– 4-sulfobenzene
Heien et al, Analyst, 2003.
Other Electrode Materials
• Other microelectrode materials– Pt– Au: harder to keep clean– BDD: decreased fouling
• Plate materials onto W– increased rigidity– bend without damage
Hermans et al, Langmuir, 2006.
Fast-scan cyclic voltammetry
i
i ?
10μm
An electrochemical method to collect current signal derived froman applied waveform, typically triangular waveform.
wavefo
rm
Carbon Fiber
Glass
Carbon Fiber
Glass
The Electrical Double Layer on a carbon-fiber microelectrode
Diffuse part of double layer (0.3-10nm)
Tightly adsorbed inner layer
i ?Capacitive charging current
Faradaic current
bulk
Capacitive charging current
iRs Cd
Circuit Model with Rs representing solution resistance and Cd for double layer capacitance at the electrode surface
A B
veCdvi
CdQdtdQRstv
EEE
CdRst
CdRsAB
∝−••=
+=•
+=
⋅−
]1[
/)/(
scan rate
Vol
tage
Time
v▪t
Capacitive charging current
curr
ent
Time
vi∝
volta
ge
Properties:
• Current amplitude is proportional to scanning rate (viz., )• Negligible e.g. 10mV/s at a regular macro-size electrode
• Substantial e.g. 1000v/s at a micro-size electrode,such as μCFE
• Induced by voltage scanningvi ∝
Model predictedRecorded from a μCFE
Faradaic current
Electroactive Solutes
dopamine
serotonin
histamine
),(),(ln
tsurfCtsurfC
nFRTE
R
oo +=
i surfxo
o xtxCnFAD =∂
∂= ]),([
E
E
i proportional to fluxFaradaic current
curr
ent
Time
volta
ge
Faradaic current
Properties:
• generated by redox couple
• 2/12/12/12/381069.2 vCvADnipeak ∝••×=
CTsVratescanvsmtcoefficiendiffusionDLmolionconcentratbulkCmareaA
o25:;)/(:;)/(:);/(:;)(:
2
2
2/1vipeak ∝
i = icharging + ifaradaic
Fast-scan cyclic voltammetry
i
i = icharging + ifaradaic
10μm
wavefo
rmMeasurable Fast-scan cyclic voltammetry also known as
background-subtracted cyclic voltammetry
i = icharging + ifaradaic
-0.4
1.0
-0.4
E
Oxidation zone
Reduction zone
0 100ms 200ms
Parameters to define a waveform pattern:
• Positive/Negative limits• Scanning rate (e.g. at a typical μCFM, it takes 10ms to finish a cycle at about 300v/s)• Applying frequency (time delay between two consecutive cycle, e.g. 10Hz shown above)
Time
i?
-0.4
1.0
-0.4
Oxidation zone
Reduction zone
0 15s
E ▪ ▪ ▪ ▪
Dopamine transients
i
-0.4 v 1.0 v
Dopamine cyclic voltammogram
t
i
Advantages:• high spatial resolution due to micro-scale electrode, particularly useful in studying exocytosis on single cell level and physiological transients of chemical messengers in slice preparation.
• high temporal resolution due to extremely high scanning rate, offering the capability to follow milli-second transient in physiological environments.
• Offering molecular identity
Disadvantages:• Sensing targets limited by electroactivity• fouling effects by cellular debris, protein etc.
Chemical messenger transients in brain slice
-5.00
10.00
5.00
0.00
1000
0
100
200
300
400
500
600
700
800
900
1500 25 50 75 100 125
Color Plot5.00
-0.50
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
14.90.0 2.0 4.0 6.0 8.0 10.0 12.0
Current (nA) vs Time (s)4.00
-2.00
-1.00
0.00
1.00
2.00
3.00
0.90-0.40 -0.20 0.00 0.20 0.40 0.60
Cyclic Voltammogram (nA vs V)
15s 15s
Following dopamine transient in a mouse brain
Electrical Stimulation
-0.4V
-0.4V
1.0V
Exocytosis in Single cells
Carbon Fiber
Glass
Carbon Fiber
Glass
-2.00
3.00
2.00
1.00
0.00
-1.00
1000
0
100
200
300
400
500
600
700
800
900
1000800 825 850 875 900 925 950 975
Color Plot
1.305
Voltage for I vs T (V)
1.6
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Time (s)29.7-0.3 5.0 10.0 15.0 20.0 25.0
Data
Stim
5s
30s
1.60
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.400.20 0.40 0.60 0.80 1.00 1.20
Cyclic Voltammogram (nA vs V)
Following single granular release of histamine from a human basophil
0.2V
1.4V
0.2V
-2.70
4.00
2.00
0.00
1000
0
100
200
300
400
500
600
700
800
900
2000 20 40 60 80 100 120 140 160 180
Color Plot
1.60
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.500.10 0.40 0.60 0.80 1.00 1.20
Cyclic Voltammogram (nA vs V)
5-HTHA
Cyclic Voltammetry AmperometryElectrode held at constant potential
Current corresponds to oxidation or reduction of analyte
• Improved time resolution
• No need for calibration
• Loss of chemical information
Single Cell Amperometry
Carbon Fiber
Glass
Carbon Fiber
Glass
i
t
StimulatingPipette
Microelectrode
+650 mVvs. Ag/AgCl
R. Mark Wightman
Rizo and Sudhof. Nature Reviews Neuroscience. 3:641-653, 2002.
Mechanism of Exocytosis
TransportTethering
Docking Priming
FusionBudding
** * **
Digital Filtering
200 Hz Filter
From Mast Cells:
Filter cut-off frequencies must be chosen carefully and vary by cell type.
Over filtering5 kHz
1 kHz
400 Hz
250 Hz
over shoot distortion
From dopaminergic neurons
Spike detection parameters:
•Amplitude threshold (5xRMS)•Area threshold •Period to find local maximum•Period to set base line•Fraction to find decay
Data output parameters:Peak maximumSpike AreaHalf-widthRise timeDecay timeSpike numberSpike frequency% of Spikes with feetFoot area / Spike Area
Data Analysis
Spike Analysis using Mini Analysis
Spike detection:Parameters:Amplitude threshold (5xRMS)Area threshold Period to find local maximumPeriod to set base lineFraction to find decay
Data output parameters:•Peak maximum•Spike Area (N = Q/nF)•Half-width•Rise time•Decay time•Spike number•Spike frequency•% of Spikes with feet•Foot area / Spike Area
NH3
HO
OH
OH
HN
OO
OO3S
SO3
OH
OSO3
HO
O
O
dopamine
heparin
Spike detection:Parameters:Amplitude threshold (5xRMS)Area threshold Period to find local maximumPeriod to set base lineFraction to find decay
Data output parameters:•Peak maximum•Spike Area (N = Q/nF)•Half-width•Rise time•Decay time•Spike number•Spike frequency•% of Spikes with feet•Foot area / Spike Area
Spike detection:Parameters:•Amplitude threshold (5xRMS)•Area threshold •Period to find local maximum•Period to set base line•Fraction to find decay
Data output parameters:•Peak maximum•Spike Area•Half-width•Rise time•Decay time•Spike number•Spike frequency•% of Spikes with feet•Foot area / Spike Area
Spike detection:Parameters:•Amplitude threshold (5xRMS)•Area threshold •Period to find local maximum•Period to set base line•Fraction to find decay
Data output parameters:•Peak maximum•Spike Area•Half-width•Rise time•Decay time•Spike number•Spike frequency•% of Spikes with feet•Foot area / Spike Area
Pre-spike foot
Biological Applications of Microelectrodes
Extracellular Measurements
Intracellular Measurements
In Vivo Measurements
Patch AmperometrySECM / AmperometryCholesterol Enzyme Electrode
Electrodes and AnalytesIntracellular Patch Electrochemistry
Release of NT’s in the braincorrelates with behavior
Extracellular Measurements: Patch AmperometryCombination of Amperometry and Electrophysiology
M. Lindau and coworkers Nature 1997 389 509-12.
Extracellular Measurements – Patch Amperometry
Pros: Best of both worlds
Simultaneous Determination of:Neurotransmitter flux (Faradaic current transient)Membrane fusion (capacitance step)Fusion pore opening (conductance change)
Investigation of:Nature of the fusion pore (evolution, size, lifetime)Kiss-and-Run eventsCoupling of vesicle size and contentIonic exchange through fusion pore
Cons: Difficult implementation
Extracellular Measurements: Electrochemical Imaging and Amperometry
3-D imaging of cellular morphology
Morphological changesconcurrent with exocytosis
J.E. Baur and coworkers Anal. Chem. 2005, 77, 1111-1117
Extracellular Measurements:Electrochemical Determination of Membrane Cholesterol
Platinum electrode with covalently attached cholesterol oxidase
Chol Oxidase
Chol Oxidase
Cholesterol + O2
Cholestenone + HOOH
HOOH + 2H+ + 2e-
2H2O
Platinummicroelectrode
James D. Burgess and coworkers JACS 2007, 129, 11352
Extracellular Measurements:Electrochemical Determination of Membrane Cholesterol
Xenopus Oocytes Mammalian Macrophages
James D. Burgess and coworkers JACS 2007, 129, 11352
Intracellular Electrochemical Measurements
Almost exclusively inside invertebrate cells
Aplysia californica Planorbis corneus
Cholinergic neuronsMetacerebral serotonergic neurons Giant dopamine neuron
Intracellular Electrochemical Measurements
Electrode Materials Intracellular Analytes
Carbon fiber (disk)Carbon ringPlatinumPlatinized carbonImmobilized enzymes on carbon or Pt
OxygenDopamineSerotoninMetronidazole (drug)Antipyrine (drug)Glucose
Intracellular Electrochemical MeasurementsCarbon ring microelectrodes
Intracellular Electrochemical Measurements
Carbon deposition by pyrolysis of methane
~1 μm tip diameter12 – 120 nm ring thickness
Ewing and coworkers Anal Chem 1986, 58, 1782
Intracellular Electrochemical MeasurementsIntracellular Patch Electrochemistry
Applicable to mammalian cells
D. Sulzer, M. Lindau and coworkers J Neurosci 2003, 23, 5835
Intracellular Electrochemical MeasurementsIntracellular Patch Electrochemistry
Voltammetry AND Amperometry
Catecholamines AND metabolites
D. Sulzer, M. Lindau and coworkers J Neurosci 2003, 23, 5835
In Vivo Electrochemical MeasurementsCorrelation with behavior
Cocaine lever pressesLever approach
Lever press
Cocaine infusion
A/V stimulus
Wightman, Carelli and coworkers Nature 2003, 422, 614.
In Vivo Electrochemical MeasurementsCorrelation with behavior
Dopamine is released after cocaine-associated stimuli only
Wightman, Carelli and coworkers Nature 2003, 422, 614.
In Vivo Electrochemical MeasurementsCorrelation with behavior
Electrically evoked dopamine release promotes cocaine seeking behavior
Wightman, Carelli and coworkers Nature 2003, 422, 614.