microelectrochemistry for chemical messenger detectiondopamine cyclic voltammogram t i. advantages:...
TRANSCRIPT
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Microelectrochemistry for Chemical Messenger Detection
Haynes Group Tutorial11/15/2007
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Tutorial Outline
• Introduction to microelectrodes• Fast-scan cyclic voltammetry• Constant potential amperometry• Bioanalytical applications of
microelectrochemistry
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Relevant Redox Reactions
NH2
OH
OH
NH2
O
O-2e-, -2H+
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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.
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+
Microelectrode Fabrication
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Microelectrode Fabrication
Cure
Polish
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AmperometryCarbon Fiber
Glass
Carbon Fiber
Glass
i
t
StimulatingPipette
Microelectrode
+650 mVvs. Ag/AgCl
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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
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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:
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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
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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
= =
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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.
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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.
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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
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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
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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
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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
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Faradaic current
Electroactive Solutes
dopamine
serotonin
histamine
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),(),(ln
tsurfCtsurfC
nFRTE
R
oo +=
i surfxo
o xtxCnFAD =∂
∂= ]),([
E
E
i proportional to fluxFaradaic current
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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 ∝
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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
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-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?
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-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
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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.
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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
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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
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-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
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Single Cell Amperometry
Carbon Fiber
Glass
Carbon Fiber
Glass
i
t
StimulatingPipette
Microelectrode
+650 mVvs. Ag/AgCl
R. Mark Wightman
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Rizo and Sudhof. Nature Reviews Neuroscience. 3:641-653, 2002.
Mechanism of Exocytosis
TransportTethering
Docking Priming
FusionBudding
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** * **
Digital Filtering
200 Hz Filter
From Mast Cells:
Filter cut-off frequencies must be chosen carefully and vary by cell type.
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Over filtering5 kHz
1 kHz
400 Hz
250 Hz
over shoot distortion
From dopaminergic neurons
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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
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Spike Analysis using Mini Analysis
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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
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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
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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
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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
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Biological Applications of Microelectrodes
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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
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Extracellular Measurements: Patch AmperometryCombination of Amperometry and Electrophysiology
M. Lindau and coworkers Nature 1997 389 509-12.
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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
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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
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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
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Extracellular Measurements:Electrochemical Determination of Membrane Cholesterol
Xenopus Oocytes Mammalian Macrophages
James D. Burgess and coworkers JACS 2007, 129, 11352
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Intracellular Electrochemical Measurements
Almost exclusively inside invertebrate cells
Aplysia californica Planorbis corneus
Cholinergic neuronsMetacerebral serotonergic neurons Giant dopamine neuron
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Intracellular Electrochemical Measurements
Electrode Materials Intracellular Analytes
Carbon fiber (disk)Carbon ringPlatinumPlatinized carbonImmobilized enzymes on carbon or Pt
OxygenDopamineSerotoninMetronidazole (drug)Antipyrine (drug)Glucose
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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
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Intracellular Electrochemical MeasurementsIntracellular Patch Electrochemistry
Applicable to mammalian cells
D. Sulzer, M. Lindau and coworkers J Neurosci 2003, 23, 5835
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Intracellular Electrochemical MeasurementsIntracellular Patch Electrochemistry
Voltammetry AND Amperometry
Catecholamines AND metabolites
D. Sulzer, M. Lindau and coworkers J Neurosci 2003, 23, 5835
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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.
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In Vivo Electrochemical MeasurementsCorrelation with behavior
Dopamine is released after cocaine-associated stimuli only
Wightman, Carelli and coworkers Nature 2003, 422, 614.
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In Vivo Electrochemical MeasurementsCorrelation with behavior
Electrically evoked dopamine release promotes cocaine seeking behavior
Wightman, Carelli and coworkers Nature 2003, 422, 614.