1

Physiological Flow Studies Group,Department of Bioengineering

6th April 2004

Electrochemical methods in physiology: from mass transport to signalling

Danny O’Hare

http://www.bg.ic.ac.uk/Research/BioSensorGroup

OverviewElectrochemical sensors- what’s worth measuring?

1. Tissue perfusion and permeability.

H2 wash-in method

Scanning electrochemical microscopy

2. Vesicular release of 5-HT.

3. New signal processing for electrochemistry.

Empirical mode decomposition and the Huang-Hilbert Transform.

Electrochemical sensorsWhy measure concentration?

• Correlation with known disease states: diabetes, drug overdose, poisoning, drug monitoring

• Legal requirements: blood alcohol, recreational substances

• Research normal and pathophysiology: nutrient supply/transport, signalling and control

So what’s worth measuring? O2, pH, perfusion, NO, glucose, lactate… e.g. solute transport in cartilage, regional ischaemia, electrical conditioning of skeletal muscle

Why use microelectrode sensors?Unparalleled and tunable spatial and temporal resolution- experimenter must make explicit decision about the length scale.

Relative independence of signal from flow.

Cheap

Continuous real time recording

More than one analyte

High rates of mass transport ⇒ fast response times, rapid attainment of steady state, detection of short-lived species

SO WHY DOESN’T EVERYBODY USE THEM?

2

Pitfalls of in vivo sensingCHEMIST: Look what your animals have done to my electrode (poisoning)

PHYSIOLOGIST: Look what your electrodes have done to my animal (and you’ve still given me no useful data)

How invasive are electrode measurements?

Rabbit tibialis anterior subjected to full tetanic contractions for 200 ms at 2 s intervals for four hours(J. Musc. Res. Cell Mot. 21 285-291 (2000)

1.Tissue perfusion- the H2 wash-in method(Greenbaum et al. J. Musc. Res. Cell Mot. 21 285 (2000)

A bolus of H2 introduced into the breathing mixture.

( ) ( )

τ−−

−=− ∞∞0

0ttexpiii)t(i

But for the bolus technique, i∞ is not known-calculate τ from the zeroth and first moments:

∫ ∫==1

0

1

0

t

t

t

t10 tdt)t(iM and dt)t(iM

( )( )( )01012

10

00121

1

iittMMttM−−−

−−=τ

Resting muscle10% duty cycle

There is no obvious relationship between perfusion and oxygenation

Measurements of dissolved H2 in paced rabbit tibialis anterior

Continuous measurement of tissue perfusion (1)

H+

H2(aq)H+

-e-Current proportional to fraction of H2 (aq) collected

H2 (aq) transported from the sensor surface by convection and diffusion

+e-

•Generate dissolved H2 by electrolysis of H2O

•Some H2 is collected by the second electrode, some is convected away

•Local generation allows measurements in poorly perfused tissue

•Continuous recording facilitates examination of the effects of pharmacological or physiological intervention

3

Sensitivity controlled by geometry

Novel thin film Pt coating on polyester-coated Pt microwire

0.7

0.6

0.5

0.4

0.3

0.2

0.1

10-

210-1 101100

α

ic/ig

on

off off off off

on on on

Preliminary in vitro data

1

22

DQL

=αwhere Q is blood flow flow per unit time per unit volume of tissue

Continuous measurement of tissue perfusion (2)

Applications

•Organ transplantation

•Stroke

•Heart disease

•Chemical process

2. Scanning electrochemical microscopy for cellular length scale measurements of tissue permeabilityThe problem: cartilage is avascular. Cells must be supplied by diffusion. Impaired mass transport has been associated with disease.

H & E stained human femoral head cartilage Bovine phalangeal cartilage

Imaging oxygen permeability in cartilageBiophys. J. 73 2771 (1997); ibid. 78 1578 (2000)

Topography, chemical imaging

Microscopic permeability- solute transport in cartilage

Bovine metacarpal phalangeal cartilage

Native PG-depleted

Topographic SECM imaging using a non-permeable tracer

4

SECM as a local probe of oxygen permeability

Approach curves for impermeable tracer (Ru(CN)6

4-)

Approach curves for oxygen

Imaging the oxygen permeability

Normalised current map Permeability map

Sub-cellular permeability is heterogenous

Interim findings

SECM permeability imaging provides new insights into mass transport on the sub cellular length scale.

Can be applied under physiological conditions.

Non-invasive, no tissue damage.

Complementary to electron microscopy and fluorescence microscopy.

3. Vesicular release of 5-HT

The biological model: CNS of the pond snail, Lymnaeastagnalis

The problem: ageing is associated with impaired cognitive function- can this be related to problems in transmission?

11 ganglia and about ~25000 neuronsNeurons are pigmentedNeurons in the CNS are large (~100 µm)Uses all three classes of neurotransmitter – gaseous, peptide and monoamine

5

3 months(young)

12 months(old)

6 months(middle-aged)

Ageing in Lymnaea• Average lifespan is ~12 months• CNS ageing in invertebrates have been shown to

be similar to mammalian systems

Lymnea CNS

CNS of Lymnaea

500 µm

500 µm1 mm

Buccalganglia

Cerebral giant cell

Serotonin levels per cell in the CNS

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Buccal ganglia Cerebral gnaglia Pedal ganglia Viscero-parietal complex

CNS regions

Con

cent

rato

in /

fmol

/cel

l

YoungMiddle AgedOld

5-HIAA levels per cell in the CNS

0

200

400

600

800

1000

1200

1 2 3 4

CNS Regions

Con

cent

ratio

n / f

mol

/cel

l

YoungMiddle AgedOld

Increased total 5-HT with ageing

Decreased 5-HIAA with ageing

Why? Impaired metabolism? Impaired re-uptake in pre-synaptic cell?

Need real-time measurements.

LC-EC Measurements of total transmitter content The electrodes

Woods metal

Silver wire

Glass capillaryCarbon fibre

30 µm or 7 µm fibres

Inlaid disc geometry

Screened construction

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Release from various sites

80

70

60

50

40

30

20

Cur

rent

/ pA

140138136134132130128Time / s

Calcium added

40

35

30

25

20

15

10

Cur

rent

/ pA

1101009080706050Time / s

Calcium added

30

25

20

15

10

Cur

rent

/ pA

12011010090807060Time / s

Calcium added

Cell bodyAxon

Synapticterminal

(i)

Vesicular Release

1000

800

600

400

200

0

Cur

rent

/ pA

32028024020016012080400Tim e / s

0 m

V

zero

cal

cium

buf

fer

30 m

M p

otas

sium

zero

cal

cium

buf

fer

30 m M potassiumCalcium buffer

30 m

M p

otas

sium

zero

cal

cium

buf

fer

6 54.8 55.0 55.2 55.4 55.6 55.8 56.0 56.2 56.4 56.6 56.8 57.0 57.2 57.4 57.6 57.8 58.0 58.2 58.4s

Distribution of vesicular events80

60

40

20

0

Freq

uenc

y

3.02.82.62.42.22.01.81.61.41.21.00.80.60.40.20.0

Peak area (pC)

7 µm carbon fiber electrode 30 µm carbon fiber electrode

Bin width = 0.006

30 µm electrode3 cells, 152 events7 µm electrode2 cells, 171 events

80

60

40

20

0

Freq

uenc

y

0.500.450.400.350.300.250.200.150.100.050.00

Peak area (pC)

Interim findings

Electrodes can measure basal release of 5-HT in intact neuronal systems

7 µm electrodes see more unitary events

Release occurs from the cell body, axon and synaptic terminal in intact systems

Vesicular release can be observed in intactneuronal systems

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4. Hilbert transform of voltammetric dataM. Arundell et al. Electrochem. Comm. 6 366-372 (2004)

Sinusoidal voltammetry offers advantages over commonly used triangular waveforms

Faradaic to non faradaic ratio is more favourable at the 2nd harmonic

Optimisation of phase and harmonic improves selectivity

from Kuhr et al. J. Electroanal. Chem. 531 119 (2002)

Kuhr used fast Fourier transform (FFT)

Similar approaches are used in a.c. voltammetry and a.c. impedance studies

The Fourier Transform requires stationary, periodic, linear signals

Electrochemical signals are always non-linear

FFT loses phase information and adds negative power to the power spectrum

Huang-Hilbert transform represents a new mathematically valid approach ideally suited to electrochemical signals (Huang et al., Proc. R. Soc. Lond. A. 454 903 (1998).)

David Hilbert (1862-1943) mathematician

At an engineering convention David Hilbert was asked not to make any jokes about their profession. He replied

“ You don’t have to worry about that. How could I possibly offend anyone? Mathematics and engineering have absolutely nothing in common”.

Timecos wt

sin wt

Imaginary Axis

Real Axis

The Hilbert transform creates the in-phase and quadraturecomponents from the analytic waveform to generate the phasor.

This generates (ii) instantaneous frequency (dθ/dt) and (ii) amplitude as a function of time.

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0 1 2 3 4 5 6 7 8-1

0

1

time/s

inpu

t sig

nal/a

rb u

nits

0 1 2 3 4 5 6 7 80

0.5

1

time/s

ampl

itude

/arb

uni

ts

0 1 2 3 4 5 6 7 80

5

10

time/sinst

anta

neou

s fre

quen

cy/H

z

(a)

(b)

(c)

0 1 2 3 4 5 6 7 8 9 100

100

200

300

400

500

600

frequency/Hz

|P|/a

rb u

nits

(d)

Comparison of Hilbert with FFT on test data

(a) Input signal

(b) Amplitude plot of (a)

(c) Instantaneous frequency plot of (a)

FFT of (a)

Application of the Hilbert transform to voltammetric data

(i) Reversible redox couple, Ru(NH3)62+/3+

5 6 7 8 9 100

0.05

0.1

0.15

time/s

ampl

itude

/mA

5 6 7 8 9 102

4

6

8

10

12

14

time/s

5 6 7 8 9 100

0.05

0.1

0.15

time/s

ampl

itude

/mA

5 6 7 8 9 102

4

6

8

10

12

14

time/s

frequ

ency

/radi

ans

s-1

5 6 7 8 9 100

0.05

0.1

0.15

time/s

ampl

itude

/mA

5 6 7 8 9 102

4

6

8

10

12

14

time/s

frequ

ency

/radi

ans

s-1

frequ

ency

/radi

ans

s-1

(a) (b)

(c)

(d) (e) (f)

Amplitude scales with concentration

Instantaneous frequency is unaltered

dθ/dt is characteristic of the electrode reaction kinetics

Application of the Hilbert transform to voltammetric data

(ii) Oxidation of p-phenylene diamine

0 1 2 3 4 5 60

5

10

15

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25

30

35

time/s

arbi

trary

uni

ts

applied potentialamplitudefrequency

10 11 12 13 14 15 160

5

10

15

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25

30

35

time/s

arbi

trary

uni

ts

(a )

(b)

Initial 6 cycles

Subsequent 6 cycles

(iii) Fouling of electrode by 5-HT oxidation products

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7-25

-20

-15

-10

-5

0

5

10

potential/V

curre

nt/u

A

(a)

21 21.5 22 22.5 23 23.5 24 24.512

14

16

18

20

time/s

ampl

itude

/uA

21 21.5 22 22.5 23 23.5 24 24.50

2

4

6

8

10

time/s

frequ

ency

/radi

ans

s-1 (e)

(d)

1 1.5 2 2.5 3 3.5 4 4.512

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time /s

ampl

itude

/uA

1 1.5 2 2.5 3 3.5 4 4.50

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4

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time/s

frequ

ency

/radi

ans

s-1

(b)

(c)

(a)

(b)

(c)

a) Voltammogram at 1 Hz

b) Initial Hilbert plots

c) After 25 cycles

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Interim findings

Hilbert transform can separate effects of concentration from the effects of electrode reaction kinetics.

Superior to background subtraction.

Can be used where background subtraction is impossible.

Preliminary results from model systems are encouraging.

CONCLUSIONS

•Microelectrodes can provide unique information on biologically relevant physico-chemical properties of living tissues.

•New signal processing approaches maximise the extraction of information from electrochemical signals and allow in situ monitoring of electrochemical sensor performance.

“…young students were employed in this labour…which however might still be improved and much expedited if the public would raise a fund...” Swift, Gulliver’s Travels, 1726

Acknowledgements

Imperial: Martin Arundell, Kim Parker, Alex Lindsay, Bhavik Patel, Costas Anastassiou.

University of Brighton: Alison Willows, Mark Yeoman

University of Warwick: Pat Unwin, Julie Macpherson

Money

EPSRC, Wellcome Trust, Royal Society