Cursul 4- Biodynamics

Impedimetric/Dielectric behavior of a suspension of non-spherical particles

Eugen Gheorghiu
Ee
A uniform electrical field, is applied to a regular distribution of
particles, i.e., the suspension is equivalent to a rectangular lattice
containing one particle (of volume Vp) in the centre of each
element.  
E0 = E 0 N
E1 Suspension permittivity, which relates the volume averages of the
displacement and electrical field on a lattice unit:
 
D =  sus E
   
 sus  E =   (r ) E (r ) dV
1
V rV
is derived using the mean field method, by taking into account the
mutual dipole-dipole interaction between all suspended particles.
The (total) electrical field is assumed to exhibit the same
distribution regardless the lattice unit emplacement and is given by:
  
E = E e + E1

E
where, e stands for the effective field, due to the external sources
(including the dipoles of the particles outside the respective lattice
unit), while E1 represents the electrical field of the induced dipole by
cell polarisation in the effective field.
   
Considering:
( sus −  O ) E =
p 1
  ( − O )E  N dV  d
V p 4 Vp 

    
P =
1
   (r ) r dS d =
1
  ( −  O )E  N dV d
4V 4V Vp 

 Vp 
P =   O    Ee
V

( 
E = 1 − 1 p    Ee
3
)
One obtains:
  O
 sus = O + p

1− p 
3
 
   (r ) r dS d
1
With: =
4V p  O E e 
R (  ) = A 0 + A 1 cos(  ) + A 2 ( cos(  ) )
2
 Effect of the Real Shape
S/V Spheroidal
1.06

1.04 REAL Shape

1.02


1 120

10 20 30 40 50 60
3

2
2
Shape
1
1 110
-4 -2 2 4 6
-1 -6 -4 -2 2 4 6
-1
-2
-2
-3

100
2
4

-6 -4 -2 2 4 6

-1 2

-2 90

Oblate -1.5 -1 -0.5 0.5 1 1.5

-2
Log n/Hz
Prolate
6 7 8 9
-4
 RED BLOOD CELLS
(d 2
− t2 ) (d 2 + t 2 )
Rx = − A  Cos 2 x + C 2 − ( A Sin 2 x) ; A =
2
; C= ;
8 8

No d (µm) t (µm) Sphere

1 7 0.8
2 7.5 0.9
3 8 1

1 2 3
 

250

225

200

175

150

125

100

Log ()
75
4 5 6 7 8 9
Aiming to describe the dielectric (1) dispersion of gap
junction connected cells we have considered the
following example:
a 0 + cos 2
r ( ) =
1 − a1 cos 
2
 Results
d1->0.9905, d2->0.9034, EC->70,SC->0.25, ES1->6, S1 ->0.001,ES2 -> 65,
S2 -> 0.008, EO -> 81, SO -> 0.374, ev -> 8.852 10-12, P->0.6
The permittivity spectra of two suspensions of high concentration
of gap junction connected cells (1) and of the related individual
cells, cells are oriented parallel to the electric field
permittivity permittivity
4000 8000

(1) 6000 (1)

3000

4000
2000

2000
1000
Log[10,w]
Log[10 ,,w] 5 6 7 8
5 6 7 8

2000 The permittivity spectra of a

1500 suspension of high concentration
1000
of gap junction connected cells
500
and of the related individual
cells, cells are oriented
5 6 7 8
perpendicular to the electric field
 The impedance modulus spectra (arbitrary units) of two
suspensions of high concentration of gap connected cells (1) and
the related individual cells, cells are oriented parallel to the
electric field
Abs Z Abs Z

8
6 (1)
6

4
4

2
2

Log w
5 6 7 8 9 10 5 6 7 8 9 10
Log w
14

12 The impedance modulus spectra (arbitrary

10
units) of a suspension of high concentration
8
6
of gap connected cells (1) and the related
4 individual cells, cells are oriented
2 perpendicular to the electric field
6 7 8 9 10 Log w
• Tissue analysis using Impedance
Spectroscopy: Revealing Alteration of
Membrane Structures during Ischemia
•Monitor
•Quantitative analysis
•Detection
•Prediction
Isch The ischemic Process

Met
Experimental aspects of using IS in characterisation of
ischemic tissues

Mod A microscopic model of the gap interconnected cells

Pro Fast algorithm to analyse and process impedance spectra

Rez Experimental results

Hepatic tissue

Myocardial tissue

Normalization Procedure

Con Conclusions
Insufficient oxygenation The Ischemic process
Reduced metabolic up take Acidification

Accumulation of metabolic residues Membrane depolarisation

Ionic inbalance
ATP

Oedema formation
Communication
Causes for closure of gap-junctions:
Plasma Membranesdecreased intracellular pH
F1.5nm Decreased energetic reserves

2-4 Increased intracellular Ca concentrations

nm

To protect neighboring cells

Connexon

Gap-junctions closure = characteristic

step in the early phase of ischemia
This study is mainly focused on quantitative
evaluation of the progression of tissue state during
ischemia, specifically in the domain of reversibility,
as it is being revealed by impedance spectroscopy
measurements.
Using the analysis of complete impedance spectra and
theoretical modeling we specifically address the
hypothesis that the closure of the gap junction is
responsible for the evolution of the measured impedance.
 5°C Command and
Thermostat Measuring
10°C cell
control Unit
15°C |Z|, 

25°C
35°C

Front-end

“pure” Ischemia

“perfused” Ischemia A B
 Isch
Met

800
Biological variability:
700
▪In homogeneity
600 ▪Anisotropy
500 ▪Dependency of tissue evolution
400 on tissue “history”
300 ▪Tissue processes and
2 3 4 5 6 7
substructures cooperativeness

150
Model
125
Program 100
75
50 Mod

25 Pro

Rez
2 3 4 5 6 7
Con
Real [Z]
800 Command and
Thermostat Meas. cell control unit
700
A, 
600
500
400
300 Front-end

2 3 4 5 6 7
Log[] -Imaginary [Z]
150
125
100
75
50
25

2 3 4 5 6 7
Log[]
1200
Cm
1000
Ri
800

600 R'e Re
400
Rg
C'm
3 4 5 6 7 8
400
Rg=10(2+0.5 m) , m = 16
300
Extra-cellular space
T
celula G.J
I
S
200
S
U
E 100

I U
3 4 5 6 7 8
~
 Schaefer
Vranceanu1991
1996 Asami 1999
Gheorghiu 1996

Microscopic model

❖Cell
Bruggeman Shape Method
– Hanai 7000

▪Presence ❖Cell orientation in relation with

of Organelles 6000 the applied field
❖Cell
▪Models pairs concentration
of cells 5000 O

▪Tubular
-6
❖Electrical
type properties
structures of extra/intra
4000 cellular fluids
3´ 10
❖Membrane capacitance 3000

-6 2000
2.5´ 10 ❖Gap junction structure
-6
1000 C
2´ 10
4 5 6 7 8 9 10
-6
1.5´ 10
a0 + cos 2 
r ( ) =
-6
1´ 10

5´ 10
-7 1 − a1  cos 2 
1 2 3 4 Phys.Med.Biol
▪ Univocal association of the closure of gap-junctions with
the characteristic behavior revealed by IS
▪ Gap-junction closure determines the dissapearance of
low frequency dispersion in permittivitty spectra, in
perfect agreement with experimental data.
▪ The experimental spectra relate with effects of multiple
processes ranging from membrane permeation to closure of
gap junctions and modified volume concentrations, taking
place in conjunction.
▪ The equivalent circuit approach becomes inefficient in
real cases, due to the pour correlation between the circuit
elements and the other (hypothetic) tissue parameters
revealed, in time, by the measured spectra.
▪ The broad experimental dispersions, characteristic for
biological tissues, impose the utilization of distributed
circuit elements
 A
Z * = LI + (1-) 
[ (i1 +) +( i    ) ]


Cole-Davidson
Jonscher Cole-Cole Havriliak-Negami
RS

RE
RP ZCPA

RI ZCPA
 Nonlinear, multi-parametric, complex fitting
algorithm

F ( ;{P}) Microscopic, phenomenologic

 
k
S =  wi' F ' exp − F ' ( ,{P}) + w"i F "exp − F "( ,{P})
2 2

i =1

F ( ,{P}) − Min[ Z exp]

Ffit ( ;{P}) =
Max[ Z exp] − Min[ Z exp]

with  in the range: 1Hz-20 MHz  has values between 1-10-8 s

▪ Data smoothing and noise reduction

Normalized frequencies ▪ Polynomial Interpolation of the data

▪ Iterative structure
 Hepatic tissue
Symmetric distribution of time constants
Homogeneous Two separate dispersions
Re[Z] 5 4 -Im[Z] 4
2500 700 5
600
2000 3
500
1500 400
1
2 300
1000 3
200
2
500 100 1

2 4 6 2 4 6
Log[f/Hz] Log[f/Hz]
-7 -9
8´ 10 7.4´ 10
120
260
-9
100 7.2´ 10
-7 240
6´ 10 -9
7´ 10
80

4´ 10
-7 C10HzA1 220
6.8´ 10
-9
C1.3 kHz
60
-9
200´ 10
6.6 A2
40 -9
2´ 10
-7 6.4
180´ 10
20 -9
6.2
160´ 10

50 50 100 100 150 150 200 200 250 250 300 300 50 100 150 200 250 300
50 100 150 200 250 300
Analysis based on phenomenological models
A1 A2
Z * = LI + +
1 + (i     1 ) 1 + (i     2 )
1−1 1− 2

-ImZ
A2
T2600 CE
2200
0.000011
500 2
2000 0.00001
400 CF
0.000009
1800
0.000008
300 1
1600
0.000007
200

1400 0.000006
100
50 100 150 200 250 300 350
50 100 150 200 250 300 350
Time [minutes] 500 1000 1500 2000
ReZ
Time [minutes]
A1 T1[s]
0.06
400

300 0.05

200 0.04

100 0.03

50 100 150 200 250 300 350 50 100 150 200 250 300 350
Time [minutes] Time [minutes]
 Myocardium
Enhanced inhomogenety
•Asymmetric Distribution
Anisotropy
•Dependency on fiber orientation
Instability in the low frequency range
•Excitable tissue
Re [Z] -Im [Z]
800 4, 5
4,5
3 150 3
600 2 125
2
1 100
400
75 1
200 50

25

2 4 6 2 4 6

Log [w /Hz] Log [w / Hz]

A1 A2
Z * = LI + +
1+ (i     )  1+ (i    
1
1−1 1
2 ) 
1− 2  2

Two dispersions
 The low frequency domain is affected by
experimental errors
400

300

Rez
200

100

3 4 5 6 7 8 9
Several experiments have shown two separate dispersions
at the beginning of the ischemic process proofing that
asymmetric dispersions must be associated with a
superposition of closely positioned distinct dispersions.
Normalizing procedure Evolution of the distribution parameter
0.4

0.375
X[ t ] − X[ t ] 0.35
max
NX =
0.325
X[ t ]
max
100 200 300 400 500 600 700
0.275

0.25
/ arb.units
Amplitude / arb. units
0.45 2
35 C
0.4
10 C 1.5
0.35
1 25 C
0.3 15 15 C
C 0.5 10 C
0.25 25
C
0
0.2

200 400 600 800 1000 1200 -0.5 0 0.5 1 1.5 2 2.5 3
Ischemia duration/ minutes Time / arb. units
 Real[Z100Hz]/
3
1800
1
1600 4
2
1400

1200

1000

500 1000 1500 2000 2500

Ischemia duration/ minutes

Real[Z100Hz] /  Real[Z100Hz]n. /arb.units
25 C
1

1400
0.8
25 C 10 C 15 C

1200 0.6

0.4
10 C
1000
0.2

100 200 300 400 500 -1 1 2 3

-0.2
Ischemia duration/minutes Normalized duration / arb. units
 The capability of protective solutions is proved by the delay in the
onset of significant cell/tissue alterations.
For a St. Thomas protective solution, mitochondria collapse
appears less than 120 minutes after the onset of ischemic
conditions and after 240 minutes significant alteration of cell
structures (membrane destruction!) take place.
Amplitude / arbitrary units.
2
2
1.5 1

1
3
4
0.5

-0.5 0 0.5 1 1.5 2 2.5

Time / arbitrary units

Paired experiments with St. Thomas protective solution (evolutions 1&2) and
HTK (evolutions 3&4) revealing better protective capabilities of the HTK
solution in connection to longer time intervals until the plateau values are reached
✓Tissue viability depends on the integrity of structural elements and on the
effective ways of communication between the cells.
✓By using a custom-made fast, noninvasive, automated, method for quantitative
analysis of impedance spectra and complex phenomenological models, we were
able to reveale membrane based microscopic processes (i.e. the closure of gap-
junctions) as characteristics of the early alterations of ischemic tissues in the phase
of reversibility.
✓Possible membrane alterations of mitochondria as revealed in experiments
performed on heart tissue awaits further clarification in conjunction to a more
detailed model taking into account the existence of extensive tubular system that
might alter the way the current flows inside the tissue.
✓Computer simulations based on microscopic as well as phenomenological
models suggest that the overall changes of impedance spectra are reflecting, on
tissue scale, the effect of complex processes, like membrane permeabilization,
closure of gap junctions and edema formation, running in parallel. Therefore, we
stress on the necessity of considering the parametrisation of the whole spectrum
for the proper analysis of the ischemic tissue.
✓ A normalizing procedure, based on the evolution of the distribution parameter
connected to H-N phenomenological model, able to provide an “internal”
reference system for the ischemic process, eliminating the requirement for control
experiments and enabling the comparison of different data sets.
✓Though performed on excised tissues the procedure developed might be the
starting point for a continuous monitoring system for clinical application.
Impedance Spectroscopy
on Biosensors
 Overview :

• Aim & Concept;

• Modeling the impedance behavior of Ab-Ag binding;

• Development of an operational impedance measurement unit suitable

for analysis of immunosensors;

• Tests on low molecular weight analytes (Gentamycin, K-casein) &

pathogens (Listeria, E. Coli & Salmonella);

• Conclusions & Open Problems

Aim: Development of a reliable and cost effective method to
reveal the presence of target analytes of various sizes

Requirements: rapid specific high sensitivity

How: combined impedance and bioaffinity approach

Impedance Measurements Bioaffinity Reactions

(differential set-up)
(Antibody-Antigen recognition)
rapid
high specificity
non-invasive (small amplitude signals)
suitable for the interfacial reaction
+ high sensitivity (ppb)
multianalyte sample measurements
mechanisms investigation

U ( j ) Ab + Ag  Ab-Ag
Z( j  ) = = Z RE ( ) + I Z IM ( )
I ( j )

Accurate, rapid detection of binding events at the

surface of modified electrodes
 The Concept: a combined bioaffinity and impedance assay

▪Monitoring the interfacial biomolecular reaction between

immobilized antibody and the antigen binding partner (the
analyte, e.g., the pathogen) using Impedance Spectroscopy.

▪ The key idea is to reveal the presence of the analyte by

investigating the dynamics of the impedance changes at the
interface between transducer and bulk during the process of
antibody-antigen binding (coupling of specific compounds to
sensor surface).
 •specific antibodies

•immobilization procedure

S
MU
E P
T P Conductive
A O matrix Ab/Ag
L R
T
 Z0
Z probe ( t ) = Z electrode + Z interface ( t ) + Z bulk ; Z probe ( 0 ) = Z electrode + + Z bulk
N

Differential Impedance
Analyzer

Zinterface
WE G RE

ZWE ( t ) − Z BK ( t ) YBK ( t ) Y 1
Z r ( t ) = = −1 = 0  −1
Z BK ( t ) YWE ( t ) Y1 n(t ) / N + 1 − n(t )   Y0
 N1  Y

1
1
 Model
• Modeling the impedance dynamics due to Ab - Ag binding  quantitative information
on the amount of different (target) analytes in solution.

• metallic Electrode

• Conductive matrix with entrapped Ab/Ag

• Equivalent impedance of an “element” of

electrolyte susceptible to be replaced by a
Ag/Ab
Electrode
•Free Ag/Ab in solution

• Equivalent impedance for a newly

bounded Ag/Ab
It is assumed that the process of Ag/Ab binding
affects only Zinterface, i.e. the impedance of the
electrode–bulk interface that “hosts” the
immobilized Ab/Ag !
 = Ka ( N1 − n ( t ) ) -Kd n ( t )
dn
dt
if K = K (V  N − n ( t ) )
a a0 V


n ( t ) = 2N1Nv V  K  N + N V  
( )
2  2 
 Kd  + N − N V 2 + 2 K d  N + N V Coth  1 Ka t  K d  + N − N V 2 + 2 K d
 Ka0 + N1 + N v V ( ) ( ) ( ) (
 Ka  1 v   )
d
 Ka  1 v  Ka  1 v  2 0  Ka0  1 v
 0  0  0
  

1− e
− K a0 (V  N − N )t
V 1

if K d / K a0 0  n ( t ) = N1
1− N e
− K a0 (V  N − N )t
(V  NV )
1 V 1
 ZWE ( t ) − Z BK ( t ) YBK ( t ) Y 1
Z r ( t ) = = −1 = 0  −1
Z BK ( t ) YWE ( t ) Y1 n(t ) / N + 1 − n(t )   Y0
 N1  Y

1
1

ZWE ( t ) − Z BK ( t ) Y0 1
Z r = =  −1
Z BK ( t ) Y1  1 − e− Ka0 (V  N V − N 1)t  
    Y
1 − e− Ka0 (V  N V − N 1)t  / 1 − N1 − K a0 (V  N − N )t 
+ 1 −    0
   (V  NV )
e 
V 1

     1 − N1 − K a0 (V  N − N )t   Y
1
  (V  NV )
e 
V 1

  

• n(t) denotes the number of Ag bounded at time t;

• NV is the volume concentration of antigens in solution;
• N1, represents the number of active Ab present at the surface of the WE;
• Y0 and Y1 are the admittances of the “element” of bulk electrolyte and the
related, newly bound Ag respectively. The ratio Y1/Y0 depends on the chosen
pair Ab-Ag, as well on the entrapment procedure .
The sign of Z is related to the ratio Y1/Y0 whether below or above unity !!
 ….When only dissociation occurs

dn
dt
(
= - K d  n− Nn )  n (t ) = e − Kd t
( N1 − Nn ) + Nn
100

80

60

40

20

10 20 30 40
Let’s have a closer look!
Besides what we want to see something else is happening:

Two complementary processes are going on: a “recognition” one due to Ag-
Ab binding and another one related to hydration of the matrix. During
hydration the dextrane layer (sensitized) has a hydrogel like behavior
(dependent of the dextrane thickness) that influences the evolutions of the
measured parameters.

With this representation in view, one can derive the time evolution of the
stabilization and recognition processes in relation to volume concentration
of the analyte, the number of active sites and values of the elements of
impedances related to each process.

1
Z channel (t ) = Z counter + + Zbulk (t )
N1 − na (t ) na (t ) N 2 − nh (t ) nh (t )
+ + +
Za Z aa Zb Zbh
This approach has the advantage that the hydration process can be
separated from the overall response (by fitting the evolutions during
stabilization, prior to injection and than subtracting this component
from the rest of the sensorgram).

Understanding the dynamics, one could decrease the measuring time,

as there is no need to wait until the sensors are fully stabilized neither
prior nor after injection. The reference electrode should ideally have the
same behavior as the working electrode regarding nonspecific binding
and hydration.

However, in reality, it is difficult to achieve this goal, therefore the effect

of hydration should be explicitly considered and corrected for prior to
deriving the differential impedance.
 Transducer development

Preparation of immuno- coated electrodes

Development of the Measuring system

Accomplishment of a dedicated impedance spectrometer module for assessing

impedance changes due to coupling of target analyte at the surface of the coated
electrode able to measure one or an array of immuno-coated electrodes.
 Differential impedance assays reveal the variations of Impedance
(capacitance & resistance) of the sensor / probe interface in response to
specific recognition events (Ag-Ab) while eliminating the nonspecific
influences (medium composition, temperature effects….).

Multi-channel, differential instrumentation

Stable sensorics

Optimized flowing conditions

-flowing chambers
-injection system

Laboratory scale
Experimental set-up:

Distinct measurement cells containing reference electrodes and active

electrodes, respectively were connected to the impedance analyzer and
successively analyzed.

Impedance PC
analyzer
Measurement
Reference Control &
Probe
Data analysis

1 2

The interaction of the imunosensor with different antigen concentrations

resulted in the formation of an antibody-antigen (Ab-Ag) complex that produced
measurable impedance changes. The dynamics of these reactions could be
observed continuously from time evolution of impedance changes. The extent
of the interaction was found to depend on the surface loading (antibody
concentration) and the time of exposure to the antigen solution!
EQUIPMENT DEVELOPMENT
✓ 4 sensors – 8 sensors (extension board)
✓ 10mHz – 150 Hz
✓ variable gain
✓ Off-set measurement /
Off-set compensation on each sensor
✓ DC voltage –200mV-+200mV applied;
option for distinct value for each sensor
✓ Actual Differential measurements
(versus user selected reference sensor)
✓ 1k – 200 k 
✓ Accuracy ±10 - 4 absolute impedance, resolution 1 m
✓ Accuracy ±0.01° phase, resolution 0.0001°
✓ Temperature measurements 0°-100 °C (accuracy ±0.1 °C,
resolution 0.01 °C)
✓ Data display:- actual data, an old data file
✓ Calibration: new or user defined file
 Front view of the multichannel differential
spectrometer developed at ICB

•high accuracy in determining the phase shift and amplitude of the measured
potential difference developed across the sample, at low currents ~ 5A
 rinsing
Impedance
|Z|/

4580

4570

4560
inj Regeneration
4550

4540

4530

4520
0 10 20 30 40 50 60 70

Time/minutes

Time
 |Zdiff|/
600
(5)
500
(4)
400
300
(3)
200
(2)
100 (1)

10 20 30 40 50 60 70
Time / minutes
The evolution of differential impedance modulus for injection of different analyte concentrations
(the arrows indicate the beginning of the rinsing step)

Experimental data
 |Z|/
Simulated data

500

400

300

200

100

10 20 30 40 50 60
[Analyte]inj / Arb units
Gentamicin detection

Differential Impedance
Direct assay
80

60
Inj
40
Competitive assay
20

5 10 15 20 25 30
-20 Time/minutes

Differential impedances for direct and competitive analysis revealing 75% inhibition of
the signal associated with A-gentamicin injection due to the presence of 100 ng/ml
gentamicin in solution
100 ng/ml ->The admissible limit of gentamicin in milk
 500

450 1:1000 A-genta

400

350

300
1:1000 A-genta

|Z| / ohm
+50 ng/mL Genta
250
1:1000 A-genta
200 +100 ng/mL Genta
150 1:1000 A-genta
+150 ng/mL Genta
100

50

-50
0 10 20 30 40 50 60
Time / minutes

Competitive assay against gentamicine

Diff impedance_Arb units

0.3
Specific
0.25
injection
0.2
0.15
0.1
0.05 Nonspecific
0 injection
0 10 20 30 40 50 60 70
Time [minutes]

Cells
Experimental data 350

300

250

200

150 10uL mAb

|Z| / ohm
100

10uL mAb+
50
2 ppm k-cas
0
10uL mAb+
3.5 ppm k-cas
-50 0 10 20 30 40 50 60 70

Time / minutes
5 Hz

Competitive assay against k-casein

Experimental data

0.5

0.4

1- |Z| mixt /  |Z| A-genta

0.3

signal atenuation
0.2

0.1

0.0

-20 0 20 40 60 80 100 120 140 160

conc Genta / ng/mL

Experimental data

0.8

0.7

0.6

1-Phasemixt/PhaseAgenta
0.5

signal atenuation
0.4

0.3

0.2

0.1

0.0

-0.1
-20 0 20 40 60 80 100 120 140 160

Genta conc / ng/mL

Cellular Platforms/cells embedded in filter
Cellular Platforms/cells coating the filter
 4000

|Z|/ Log(n)
0
0 1 2 3 4 5

-10
3000

-20

2000
-30

-40

1000

-50

-60

0 1 2 3 4 5
Log(n)
-70

Experimental data |Zdiff|/
500

400

300

200

2 3 4 5 6 7 8
5
Cell concentration x 10 /ml
Calibration curve for injection of different pathogen (Listeria monocytogenes) concentrations:
from 1.6 x 105 to 8 x 105 cells/ml