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.