Electrochemical impedance spectroscopy (EIS)

ELECTROCHEMICAL IMPEDANCE
SPECTROSCOPY

COLLEGE OF APPLIED SCIENCES
Department of Industrial chemistry
Analytical chemistry
Electrochemical impedance spectroscopy (EIS) in biosensing
By: Getasile Assefa
getasile.assefa@aastu.edu.et
getasile@gmail.com
Addis Ababa science and technology university April, 2019
ADDIS ABABA, ETHIOPIA
21-Jun-19 2
ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY (EIS) IN BIOSENSING

Introduction
Basic principle of electrochemical impedance spectroscopy
Impedance biosensors
Application of electrochemical impedance spectroscopy
Current trends and challenges
Conclusion 21-Jun-19Electrochemical impedance spectroscopy (EIS) in biosensing 3

ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY (EIS)
 EIS is a powerful method of analyzing the complex electrical resistance of a system ( is sensitive
to surface phenomena and changes of bulk properties)
 It can be used to determine semi-quantitative parameters of electrochemical processes occurring
at electrode surfaces
………. EIS provides a fingerprint of the interfacial region (Maalouf et al. 2007).
21-Jun-19Electrochemical impedance spectroscopy (EIS) in biosensing 4
 First examples of its use was reported at the end of the 1980s;
however, the method has seen a huge improvement in recent years
due to;
 Advances made in instrumentation and
 Its extraordinary sensitivity
Introduction
Fig. Kinetic processes taking place
at electrode-electrolyte interface

…It has been applied for studying electrochemical
properties, for example,
 Charge transport across membranes & its
interfaces
 Electrode kinetics,
 Double layers studies
EIS has been intensively used in areas,
Electrochemistry,
Bio- medical applications ,
 Material science and others
Bio-sensing ,
Energy storing and conversion systems
(Fuel cells, Rechargeable batteries)
Corrosion mechanisms and
Electrochemical synthesis, 21-Jun-19Electrochemical impedance spectroscopy (EIS) in biosensing 5
Cont..
Energy Environ. Sci., 2009, 2, 215–219
A. Manickam et al., Sensors 2012, 12, 14467-14488

In the field of biosensors, it is particularly well-
suited for the detection of binding events on the
electrode surface for a wide range of chemical and
biological targets
Besides the detection of biorecognition processes, it
is a valuable tool for characterising of surface
modifications,
J-Y. Park & S-M.Park, Sensors 2009, 9, 9513-9532
…Due to change of electrochemical properties
of electrode interfaces during immobilisation of
biomolecules on the transducer

Advantages
1. Useful on high resistance materials
2. Time dependent data is available
3. Non- destructive
4. Quantitative data available, etc
Disadvantages
1. Expensive
2. Complex data analysis for
quantification

Making EIS Measurements
I. Apply a small potential (or current) AC signals (1 – 10 mV) of fixed
frequency or for a wide range of frequencies.
II. Measure the response and compute the impedance at each frequency.
III. Plot and
IV. Analyze
Voltage
1 Cycle
Alternating current (Ac)
Basic principle of electrochemical impedance

SINUSOIDAL CURRENT RESPONSE IN A LINEAR SYSTEM
The excitation signal…
0( ) cos( )E t E t
0( ) cos( )I t I t  
The Response signal…
0
0
0
cos( )( ) cos( )
( )
( ) cos( ) cos( )
E tE t t
Z t Z
I t I t t
 
   
  
 
The impedance is therefore expressed in terms of a magnitude, Z0,
and a phase shift, ɵ . It may allso be written as complex function:
 IZI is composed of a real and an imaginary part
 Impedance is represented as vector with length IZI
Figure. Phase shift in current
I(t) as a response to excitation-
potential E(t) in a linear
system

Nyquist plot
0
10
20
30
40
50
60
70
80
90
100
-3 -2 -1 0 1 2 3 4 5 6 7

log f
Bode plots
Bode plot
Nyquist
Representations of EIS
 = 1/RctCd
Kinetic control
ZIm
ZRe
Rs Rs+ Rct
Mass-transfer
control

 Models and analogs interpretation
 Analogs which always take the form of electrical equivalant circuits models
Analyzing EIS: Modeling
Analyzing EIS: Modeling
oElectrochemical cells can be modeled as a network of passive electrical circuit elements.
oA network is called an “equivalent electrical circuit”.
…can be done using serial and parallel combinations of Circuit Elements
oMost of the circuit elements in the model are common electrical elements such as,
Double Layer Capacitance (Cdl)
Electron Transfer Resistance (Ret)
Uncompensated (electrolyte) Resistance (Rs)
Warburg impedance (zw):

Double Layer
Capacitance (Cdl)
Electron Transfer
Resistance (Ret)
Uncompensated
(electrolyte)
Resistance (Rs)
Randles Cell
(Simplified)
Randles Cell

oThus in Nyquist plots, EIS data is analyzed by fitting it to an equivalent
electrical circuit model
(Dot and solid lines, in fig. ).
J. Nanosci. Nanotechnol. 15, 3385–3393, 2015
Figure, Correlation between Nyquist plots for:
(A) Bare GCE;
(B) SWCNT modified GCE;
Thus, each response of an equivalent
circuit in EIS measurement can be
calculated and compared to the actual
response of the electrochemical cell.
Additionally, by using the plots we
can calculate values of the elements
of the equivalent circuit for any
applied bias voltage
In order to verify the validity of the chosen circuit, the quality
of the fit to the experimental curve must be evaluated…….

Impedance biosensors
 Like all electrochemical biosensors, impedimetric sensors are
bio-electronic devices that make use of the interactions of
biomolecules with a modified electrodes surface.
 The detection process involves the formation of a recognition
complex between the sensing biomolecule (Bioreceptors) and
target analyte at the interface of transducer, which directly
or indirectly changes the electrical properties of the
electrode-solution interface
Alternatively, if the impedance or capacitance of the interface
changes when the target analyte is captured by the probe, EIS
can be used to detect that impedance change.
This is in fact, when a target biomolecule interacts with a
probe-functionalized surface, changes in the electrode-solution
interfacial properties (resistance, capacitance or CPE, etc,) can
result due to the presence of the target molecule.
EIS can be successfully applied for;
i. Characterization of electrochemical phenomena occurred at
biosensing surfaces and
ii. Evaluation of bioanalytical signals generated by biosensors
during biosensor fabrication process
phenomena at electrode-solution interface :
 Kinetics of antigen-antibody interactions,
 Redox reactions,
 Other molecular interactions

 Furthermore, because of the affordability and availability of
impedance instrumentation, currently trends towards the
development of impedimetric biosensors appears to be very
popular.
 This can be seen from the considerable progress on this topic over
the past few years .
 Thus EIS having a special interest in bioanalysis, it become a vital tool
for the detection of a wide range of chemical and biological analytes
Applications have been demonstrated for various types of (bio)analytes,
such as:
o Proteins,
o Nucleic acids,
o Whole cells,
o Microorganisms,
o Antibodies and
o Antigens

 Practically, the method of impedance biosensing is mainly
based on the interactions between the bioreceptors
(Prove) and the target analytes selectively adsorbed from
the solution.
 As a result, such interactions cause a change on interfacial
electron transfer kinetics between a probe in solution and
the conducting electrode surface.
 This electrochemical change is then detectable by
monitoring the charge-transfer resistance (Rct)
 Thus, impedimetric biosensors (Faradaic) detect
biorecognition events which occur at the modified
electrode by measuring the change in the electron
transfer resistance, Rct due to;
 Steric hindrance caused by the immobilized biomolecular
interaction or
 Electrostatic repulsion between the free charges of the
target molecules and the electroactive species in the
supporting electrolyte
Figure . Schematic diagram for an
electrode/electrolyte interface in a faradaic
sensor and its exemplary model circuit

On the other hand, EIS can be used to investigate capacitance properties made in the absence of redox
prove, occurred due to the influence of the electrical field on the biological recognition event,
In this technique, the signal of non-Faradaic method is mainly due to capacitance changes on the interface
that can be easily monitored by double-layer capacitance (Cdl) means.
The capacitance of the electrochemical double layer, Cdl, depends on all of the compounds present at the
interface, which are;
Predominantly solvent molecules,
Immobilized biomolecules, and
films
In impedance biosensing, capacitance change is brought when the
dielectric constant or the thickness of the double layer on the electrode
surface changes
Thus, EIS can be successfully applied for the characterization of
biosensing surfaces
NOTE:
Capacitors in EIS often do not behave ideally. Instead, they act like a constant phase element (CPE)

 The impedance parameter of interest (e.g. resistance or
capacitance) can be obtained from a fit of the measured
impedance data with a verified (Actual) equivalent
circuit (e.g. in figure ) ,
As a result, the overall impedance can be directly
correlated to for the
Characterization of biosensing surfaces and
Evaluation of bio-analytical signals generated by
biosensors
J. Nanosci. Nanotechnol. 15, 3385–
3393, 2015
Figure, Nyquist plots for equivalent
circuit : Bare GCE;
 Generally in impedance biosensing each process occurring
in the interface of electrode-electrolyte can be modeled to
equivalent circuits using combination of resistors (Rs), and
capacitors (Cdl)

Why Study Impedance Biosensors?
Low cost, small instrument size, and speed of analysis are crucial,
Point-of-care diagnostics – a measurement and diagnosis at a bedside, in
an ambulance, or during a clinic visit – are a promising application
Other applications include biological warfare detection, consumer test
kits, bio-process monitoring, and water quality testing
Another potential application is the label-free determination

EIS Instrumentation
 Today, most EIS investigations are carried out using dedicated lab instruments
featuring high accuracy, a wide range of test frequencies, the possibility to make
measurements with a two-, three- or four-electrode configuration in both potentiostat and
galvanostat operation mode
 EIS analysers are potentiotat designed especially for measuring AC impedance, and have
typical frequency ranges of 10 MHz – 100 kHz
Several instruments based on small-signal DC admittance measurements such as:
 LCR-meters impedance analyzers,
 Lock-in amplifiers and
 Frequency response analyzers (FRA)
Additionally, these instruments normally assembled with exclusive software for data
analysis and fitting programmes

Applications of EIS
The application of EIS has increased radically in the past few years due to its ability to elucidate both
physical and electronic properties of electrochemical systems such as;
‡ Corrosion mechanisms
‡ Adsorption and desorption to electrode surface
‡ Electrochemical synthesis of materials
‡ Catalytic reaction kinetics
‡ Label free detection sensors
‡ Ions mobility in energy transfer & storage devices; such as
‡ Batteries
‡ Supercapacitors, and
‡ Fuel cells,
Energy Environ. Sci., 2009, 2, 215–219

Mostly, EIS becomes a sensitive technique for the analysis of the interfacial
properties related to biorecognition events, like;
 Reactions catalyzed by enzymes,
Biomolecular recognition events occurring at the electrode surface by,
 Proteins,
 Antibodies or antibody-related substances,
 Lectins,
 Receptors,
 Nucleic acids,
 Whole cells,
Many studies on impedimetric biosensors are focused on immunosensors
and aptasensors (Aptamer based biosensor)

Impedimetric immunosensors
It is a sensitive technique for label-free detection of antigen –
antibody binding
It is based on measurements of electrochemical faradaic
impedance in the presence of Fe(CN)6]3-/4- as a redox probe
In impedimetric immunosensors antibodies are immobilized on
the electrodes surface following the antigens is bound to form
immunocomplex
After immunocomplex formation, it create a barrier that prevents
(hinders) the redox probe from making contact with the electrode
surface
This results impedance changes due to electron transfer
resistance (Rct) changes that can be measured with the potentiotat
instrument.
Finally, the changes in electrochemical impedance can be
correlated to specific concentrations of target analyte
Figure, Impedimetric immunosensors
Figure . Principle of the immunosensor
a) Bare ; (b) Electrode with antibody
immobilization; and (c) antibody bind with
Antigen

CASE STUDIES-1
Label-free impedimetric immunosensor based on one-step co-
electrodeposited poly-(pyrrole-co-pyrrole-1-propionic acid) and
reduced graphene oxide polymer modified layer for the
determination of melamine

Recently, Y. Gu et al. (2019) developed a label-free impedimetric immunosensor based
on a novel polymer layer for the sensitive and selective detection of melamine (MEL) in
dairy samples.
The poly (pyrrole-co-pyrrole-1- propionic acid) and reduced graphene oxide polymer
layer (rGO-PPYPPA) were co-electrodeposited on the electrode using a one-pot method,
thus obtaining a copolymer layer with favorable electrochemical properties.
The modified copolymer layer improved the conductivity of the sensor and provided
suitable active sites for antibody binding, thus enhancing immuno-binding.
 The antibody was anchored on the modified
electrode by covalent coupling.
 Then, the target & coating antigen were
added simultaneously to compete for the
capture sites of the antibody.
Scheme, preparation process and
detection strategy of impedance
immunosensor.

Figure shows characteristic Nyquist plot and CV curve of the various modified
electrodes
o The Rct value in the Nyquist plot of the modified electrode was 44.9 Ω.
o The peak current in the CV curve of the modified electrode was 382.5 μA
o After the antibody was attached to the modified electrode, an increase in Rct (360 Ω)
and a decrease in peak current (220 μA) was observed.
o This was due to the hindering eﬀect of insulating protein for electron transfer.
o Then, the blocking process and immuno-competition process further protect electron
transfer of the electrodes.
o Therefore, the Rct increased to 568 Ω after blocking and 806 Ω after immuno-binding.
Further proving from CV peak, the current decreased to 157 μA after blocking and
decreased to 108 μA after immuno-binding.
Figure. EIS & CV of (a) rGO-PPYPPA/GCE, (b) Ab/rGO-PPYPPA/GCE,
(c) EA/Ab/rGO-PPYPPA/GCE, (d) MEL-BSA/EA/Ab/Rgo PPYPPA/GCE,
Detection strategy of impedance immunosensor
The stepwise increase in impedance value confirm the successful
immobilization of GCE surface

Generally, the specific immune-interactions at the
electrode surface that restrict the electron transfer of the
redox probe lead to changes in electrochemical impedance
Thus, such changes in impedance in terms of increase in
Ret is correlated to series of MEL concentrations in EIS
analysis.
The Nyquist plot shows a linear relationship b/n the Ret
and increasing MEL concentrations
From the calibration curve, it shows a wide detection range
(10−11−10−2 mol L−1 ) and low limit of detection (1.2 ×
10−11 mol L−1) under the optimal conditions
Figure (A) EIS responses of a series concentration of targets
(10−11−10−2 mol L−1) of MEL in the electrolyte of 5 mM Fe(CN)6
3−/4
(B) Calibration curve
Calibration curve

Aptamer-based impedimetric
biosensors
‡An Aptamers is a artificial single-stranded DNA/RNA oligonucleotides that binds to a target molecules
‡Short 15-75 bases
‡They are chosen by an in-vitro selection process so called SELEX (Systematic Evolution of Ligands by
EXponential enrichment), that identifies a monomer sequence and strongly binds the target from a large library
of random sequences
‡An Aptamer does not bind to target by cannonical base-pairing like oligonucleotides used in PCR
‡Aptamer often base pair internally, making stems and loops that position bases in optimal location
SELEX on DNA
1. ssDNA Library
2. Partition in to binding/
Non-binding fractions
3. Elute binding ssDNA
4. Amplify DNA by PCR
Proteins,
Small molecules,
Cells,
Viruses /bacteria, and
Amino acids
Trends in Analytical Chemistry, Vol. 27, No. 2, 2008

Aptamers are considered advantageous alternatives to antibodies for capture
probes because of some superior features such as:
o Facile production,
o High specificity of binding affinity,
o Better stabilization/longer shelf life
o Well understood tethering chemistry, and
o Reduced cross-reactivity
In impedimetric techniques based on aptamer, changes in current, resistance or
impedance following the binding of target sequences (hybridization),
conformational changes, etc can be monitored.
After target binding a measurable change occurs with these modes:
(1) Direct detection of hybridization (label-free),
(2) Labeling of the target nucleic acid sequences with redox active substances,
nanoparticles
(3) Signal probes (indirect labels e.g. using sandwich assay) that intercalate
within the stacked base pairs, electrostatically bind to the phosphate backbone
or sit within the channels of the double helix.
Aptamers Vs Antigens

CASE STUDIES-2
An aptamer-based biosensor for detection of doxorubicin
by electrochemical impedance spectroscopy

Bahner N. et al (2017) developed aptamer-based biosensor for or the detection
of doxorubicin using impedance spectroscopy.
The principle is based on the inhibition of electron transfer between
electrode and ferro-/ferricyanide (as a redox prove) in solution caused by the
binding of doxorubicin to the immobilized aptamer (fig).
21-Jun-19Electrochemical impedance spectroscopy (EIS) in biosensing 31Figure , Immobilization and measurement principle
Thus, the binding of doxorubicin to the
immobilized aptamer can be detected by change in
electrical impedance
Daunorubicin binding aptamer as biological recognition element

 The change in impedance during the immobilization of the
DRN-aptamer was investigated by means of
electrochemical impedance spectroscopy (EIS).
 Upon binding of doxorubicin to the immobilized aptamer,
the impedance increases due to the hindrance of electron
transfer between electrode, and the redox probe of ferri-
/ferrocyanide [Fe(CN)6]3-/4-.
 Fig.12 shows a comparison among, the impedance
spectrum of a
 Bare (black cross),
DRN-aptamer-modified gold electrode (red plus) and
MCH-coated gold electrode (blue triangles)
Figure 12, Nyquist plot of a blank (black cross) electrode, a DRN-aptamer-modified electrode (red plus),
and MCH-modified electrode (blue triangles) measured in FeBB – the colored solid lines represent the fits
to the equivalent circuit
 daunorubicin-binding aptamer (DRN-aptamer)
 6-Mercapto-1-hexanol (MCH, 99%

Furthermore, to extract the relevant parameters mentioned in , Table the
impedance spectra were fitted to the modified Randles circuit (Fig. 13, inset.
Table 1 Fit parameters of different modified electrodes; Rs = solution resistance, Ceff = effective capacitance
(calculated by, Rct = charge-transfer resistance and ZW = Warburg impedance (from three different electrodes)

Finally, upon proving its analytical performance a
linear relationship between the charge transfer resistance
(Rct) and the doxorubicin concentration was obtained for
different concentrations of doxorubicin.
 Figure shows the Nyquist plot of a DRN-aptamer
modified electrode and the same after exposure to 8,
62, and 125 nM doxorubicin.
 This results, a linear relationship between the Rct and
the doxorubicin concentration with a detection limit of
28 nM.
Advantages
High sensitivity,
Selectivity, and
Simple sensor construction,
Shows a high potential of impedimetric aptasensors in for
environmental monitoring.
Figure 13, Nyquist plot of a DRN-aptamer-modified
electrode (black cross) and the same after exposure
to 8 nM (red squares), 62 nM (blue pluses), and 125
nM doxorubicin (green triangles)

Current trends and challenges
EIS has been used since decades in non-biological applications such as in
 Corrosion study,
 Electrochemical activity of Li-ion cells or
 Monitoring fuel cell performance, etc
However, EIS has gained recently much more popularity in biosensor applications
that is used for a wide range of analytes owing to its
o Label-free measurements,
o Non-destructive technique, and
o Excellent sensitivities, sometimes reaching down to the femto molar ranges

 Although EIS having the potential for direct binding detection in biosensor, many
interfacial impedimetric assays suffer from the disadvantage that the generated
signals (changes in Cdl and/or Rct) are relatively small.
 In addition to profit from the development of nanotechnology and molecular
biology, diverse fabrication and signal amplification strategies have been designed
for detection of analytes, which has led to great achievements in fast quantitative
and simultaneous testing with extremely high sensitivity and specificity .
 This has led to the development of several amplification protocols used (label-free,
enzyme labels, conducting polymer films, nanoparticles, etc.)

Conclusion
EIS technique has demonstrated its feasibility on the characterization of modified-surface electrode processes
and determining diffusion kinetics, mass-transport parameters at electrodes interfaces.
Currently, EIS has been intensively used for the elucidation of corrosion mechanisms, characterisation of
charge transport across membranes and membrane/solution interfaces of batteries.
Mainly, (EIS) is becoming a sensitive technique for the analysis of the interfacial properties related to
biomolecular recognition events on the transducer surface.
The detection process involves the formation of a recognition complex between the sensing biomolecule and
the analyte at the interface of the electronic transducer, which directly or indirectly changes the
electrochemical impedance properties of the recognition surface.
These changes due to immobilization can be measured and displayed with impedance instrumentation and
software for qualitative analysis ,
The foremost advantages of working with EIS based biosensors is the small amplitude perturbation from
steady state, which makes it a non-destructive technique, as well as its label-free measurements and excellent
sensitivities.
Also, because of the affordability and availability of impedance instrumentation at the present time, a trend
towards the development of impedimetric biosensors appears to be popular.
These can be realized from the increase in the number of publications on this topic over the past few years.21-Jun-19Electrochemical impedance spectroscopy (EIS) in biosensing 37

Electrochemical impedance spectroscopy (EIS)

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