INTRODUCTION

Impedance Spectroscopy
 Impedance spectroscopy is a method of characterizing electrical properties of materials
by applying low voltage electric field and measuring electrical properties in terms of
dielectric and conductance.
 It is a fast, easy and scalable method to investigate various systems like corrosion
performance of coated metals by measuring the frequency dependent capacitance in
time, reaction kinetics etc. [1-2].
 It is a highly sensitive and non-intrusive technique which makes it ideal for bio-sensing
and to estimate the cell growth in bioreactor [3-4].
 Impedance spectroscopy is used to understand the dynamics of mobile and bound
charges in colloidal suspensions. When the particles are suspended in aqueous medium,
an electric double layer develops around the surface of the particles.
 Under the influence of an alternating electric field, an induced electrical dipole is
formed due the frequency-dependent field-driven charge polarization of the electric
double layer.
 The impedance spectroscopy records the conductance, complex impedance,
inductance, relaxation frequency and phase angle which depends upon the particle
charge, size, nature and medium.
 All these factors are very important for determination of zeta potential or stern layer
conductance of the particle and thus, serve as a good method for characterization of
colloidal properties [5-8].

Why frequency Spectrum of Permittivity?

 Frequency spectrum of permittivity, that is, the dielectric dispersion, of a colloidal

suspension is a macroscopic manifestation of polarizability of the particles, which is
inclusive of the solid core of the charged particle along with its diffuse ionic
atmosphere.
 Factors such as particle size, shape, zeta potential, and concentration and mobility of
ions in the medium cooperatively affect the electrical state of the solid/liquid interface,
and thereby suspension permittivity.
  Theoretical models for electrokinetic response of a particle in the infinite dilution limit,
that is, where particle−particle interaction effects are not important, are well
established.1−5
 These models show a quantitative comparison between theory and experiments after
considering a finite mobility of ions in the dense part of the double layer and/or
correcting for the effect of electrode polarization at low frequencies.6−11
 Recent experimental work and theoretical development has focused on furthering the
understanding of dielectric dispersion of concentrated suspensions, which are of
increased practical and technological importance.12
 The data on complex impedance of colloidal suspensions are typically reported in terms
of dielectric and conductivity increments (with respect to medium).

zwitterionic sulphonic acid buffers Used

 N-(2-Hydroxyethyl)-Piperazine-N-2-Ethanesulphonic acid (HEPES),
 Piperazine-1,4-bis-2-ethanesulphonic acid (PIPES),
 Piperazine-1,4-bis-2-hydroxypropanesulphonic acid (POPSO), and
 4-(2-hydroxyethyl)-piperazinepropanesulphonic (EPPS)

These buffers are widely used in research for biological colloids and offer distinct aspect than other
buffers due to their zwitterionic nature. Most of the theoretical research has been performed on de-
ionized (DI) water or electrolytic media like NaCl or KCL[5-7]

Zwitterion

 In chemistry zwitterion is known as dipolar ion. Zwitterion molecule contains two or

more function groups of which one has a positive charge and the other has negative
charge and the net charge on the molecule is zero.
 The pH at which this happens is known as isoelectric point.
 The charge on the zwitterion comes from the transfer of a hydrogen ion to the amine
group.
 Since the zwitterion has capability to act as a proton donor and proton acceptor, this
naturally act a buffer.

Impedance Change (ΔZ)

The quantity that we measure in experiments is impedance while most of the theoretical work
reported in literature is in terms of complex conductivity and permittivity. This is because
complex conductivity and permittivity of particles can be expressed linearly at dilute
concentration of particle and particle-particle interactions can be ignored. The real and complex
part of the conductivity and permittivity with change in quantities associated to single particle
[7]
are given as
𝐾𝑠 (𝜔)− 𝐾𝑒
𝛥𝐾𝑠 = (eq.2.1)
𝛷

𝜀𝑠∗ (𝜔)− 𝜀𝑒∗ (𝜔)

𝛥𝜀𝑠∗ = (eq.2.2)
𝛷

Where 𝛥𝐾𝑠 and 𝛥𝜀𝑠∗ are the change in conductivity and permittivity due to single particle.

The change due to single particle can be scaled by multiplying with the volume fraction Φ (Φ
= npVp, np is the number density of particles and Vp is the particle volume) and it is given as

𝐾𝑠 (𝜔) = 𝐾𝑒 + 𝛷𝛥𝐾𝑠 (eq.2.3)

𝜀𝑠′ (𝜔) = 𝜀𝑜 + 𝛷𝛥𝜀𝑠′ (𝜔) (eq.2.4)

𝜀𝑠" (𝜔) = 𝛷𝛥𝜀𝑠" (𝜔) (eq.2.5)

The complex conductance of colloidal suspensions is related to experimentally measure

impedance given by Mangeldorf and White [5] as

1
𝐾 ∗ (𝜔) = (eq.2.6)
𝐶𝑐 𝑍(𝜔)

Where Cc is cell constant in meter-1, the method of obtaining which is given in Appendix A.2.
The real part of conductivity is a measure of resistance of the suspension and it is related as

𝐾𝑠∗ (𝜔) = 𝐾𝑠 (𝜔) + 𝑖𝜔𝜀𝑜 𝜀𝑠′ (𝜔) (eq.2.7)

Where 𝜀′𝑠 (𝜔), is the real part of the permittivity is a measure of capacitance of the suspensions and
complex permittivity is related to complex conductivity [9] as

𝐾𝑠∗ (𝜔)
𝜀𝑠∗ (𝜔) = (eq.2.8)
𝑖𝜔𝜀𝑜

For the complex conductivity of the suspending buffers [9] is related as

𝐾𝑒∗ (𝜔) = 𝐾𝑒 + 𝑖𝜔𝜀𝑜 𝜀𝑒 (eq.2.9)

Where Ke is ohmic conductivity and εe is the permittivity of electrolyte. Ke is equal to principle

conductivity which is obtained using DC conductivity experiment. It is assumed that the ohmic
conductivity and permittivity are independent of frequency.

Electrode polarization
 The measurement of impedance can be difficult due to several unwanted effects.
 One type of effect comes from non-ideality of experimental setup because the setup
consists of electrical circuits, wires and external components. It can give rise to “stray
impedance” which is accounted for in our measurement.
 Another type of effect comes from the distribution of the charged ions or molecules
within the measurement cell due to the presence of electric field that we applied. It is
known as electrode polarization which typically occurs at lower frequency (<100 kHz).
 Electrode polarization occurs due to the fact that at low frequencies, there is a charge
build up or accumulation of ions taking place at the electrode and electrolyte interface.
 This accumulation of charged ions has a major contribution towards the capacitance
during the impedance measurements and this is the reason for the high relative
permittivity at low frequencies and it makes the experimental measurements difficult.
 It is assumed that the electrode polarization is purely capacitive and the impedance of
the suspension is the sum of true impedance (in absence of electrode polarization) and
impedance due to electrode polarization [8].
Figure: Schematic of distribution of charged ion or molecule and formation of electrode double
layer at electrode surface when AC electric field is applied to suspension.

[11]
The equivalent electrical circuit for the suspension/electrolyte along with electrode
polarization is given by

Figure: Equivalent electric circuit representing bulk suspension and electrode polarization.

The effective impedance of the circuit can be written as

Z = Zs + ZEP (eq.2.10)

1
Z = Zs + (eq.2.12)
𝑖𝜔𝐶𝐸𝑃

1
Zs = + iωCb (eq.2.13)
𝑅𝑏

Where, Zs is the true impedance of the suspension and ZEP is the impedance due to electrode
polarization.
 Methodology
Materials.
1. Carboxylate-modified 1 μm polystyrene (PS) beads with 0.16 C/m2 surface charge
density (IDC Invitrogen, USA);
2. acetone and tannic acid were from Fisher-scientific;
3. sodium citrate, chloroauric acid (HAuCl4·3H2O),
4. silver enhancement kit
5. silicone grease was from Metroark;
6. polydimethylsiloxane (PDMS) and curing agent (Sylgard 184,U.S.);
7. phosphate buffered saline (PBS) tablets were from Omnipure;
8. Acetone and IPA (Fisher-scientific);
9. N-(2-Hydroxyethyl)-Piperazine-N-2-Ethanesulphonic acid (HEPES) (Sisco Research
Laboratories) or ( Sigma-Aldrich);
10. Piperazine-1,4-bis-2-ethanesulphonic acid (PIPES)
11. Piperazine-1,4-bis-2-hydroxypropanesulphonic acid (POPSO)
12. 4-(2-hydroxyethyl)-piperazinepropanesulphonic (EPPS)
13. Polydimethylsiloxane (PDMS) and curing agent ( Sylgard 184, USA);
14. Ultrapure deionized (DI) water (~ 18.2 Mohm.cm) (Millipore,India).

PDMS chip preparation.

1. PDMS and curing agents were mixed in the 1:10 v/v ratio until small bubbles are
formed.
2. To remove the small bubbles, it was degassed under vacuum for 15-30 min.
3. The glass petri dish was cleansed with IPA solution, Acetone and DI water and excess
water was removed using nitrogen gas.
4. After degassing, 10mL degassed mixture was poured slowly so that no air bubbles were
formed onto the petri dish and bake it at 650C for 4hr.
5. The solidified film was then taken out and glued to a scotch tape.

Sample Preparation.
Buffer:
1. 1× PBS buffer at pH 7.4 (containing 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate
buffer) was prepared by dissolving one PBS tablet in 100 mL of DI water.
 2. Similarly, 40 mM HEPES buffer at pH 5.3 was prepared by reconstituting 3.8 g of
HEPES powder in 400 mL of DI water.
3. The HEPES pH was further adjusted to 8.5 by adding approximately 14 mL of 1 M
NaOH. The buffer conductivities were measured using the Mettler Toledo Lab-731
ISM conductivity meter, and the pH data were collected using the Eutech Instruments
pH-7200 meter.
4. 40 mM HEPES, PIPES, EPPS and POPSO were prepared by dissolving the respective
salts into 40 ml of milli-Q water.
5. The pH of the buffers was then set at 8.5, 8.44, 8.44 and 10.84 respectively by adding
8N NaOH.

Latex particle: 200 μL of 4% w/v stock PS suspension (dia. 1 μm) was suspended in 800 μL
of HEPES buffer and was washed twice via centrifugation at 10,000 rpm for 10 min each to
remove any surfactants, electrolytes or preservatives from the medium.
 The final pellet was re-suspended in 40 mM HEPES buffer at pH 8.5 and further diluted
to different concentrations down to 0.001% w/v.
 Similarly, the particles were re-suspended in PIPES, EPPS and POPSO buffers.

Au NPs.:
1. Citrate-capped Au NPs of approximately 16 nm diameter were synthesized using the
well-known Turkevich method.22
2. Briefly, 5mL of 1% w/v HAuCl4 was mixed with 395 mL of DI water.
3. Simultaneously, a reducing solution was prepared by mixing 20 mL of 1% w/v sodium
citrate, 50 μL of 1% w/v tannic acid, and 80 mL of DI water.
4. The gold and the reducing solutions were then heated to 60 °C separately and mixed
together under constant stirring at 60 °C for 4 h.
5. Once the solution turned wine red color indicating the formation of AuNPs, the
suspension was quenched in an ice-bath and stored at 4°C. The Au NPs were then
centrifuged from 3000g to 12 000g at intervals of 3000g for 15 min each and
resuspended each time in HEPES.
6. The final concentrations were adjusted as desired by further dilution in HEPES.

Metallodielectric Particles.
1. To prepare Au NP-coated PS particles, 20 μL of 2% w/v PS suspension was incubated
with 1 mL of 1.12 nM
 2. Au NP suspension at room temperature for different time durations under continuous
shaking at 50 rpm.
3. Excess gold was removed by two successive washings at 10 375g for 10 min each, and
the final Au-PS pellet was resuspended in HEPES.
4. These Au-PS particles were further deposited with silver metal by preferential silver
ion reduction on gold.
5. For this, 20 μL of Au-PS suspension was incubated with 50 μL of silver enhancement
solution for different time durations.
6. Finally, the particles were washed thrice at 3100g for 15 min each and resuspended in
HEPES.
Sample Characterization.
1. The particles were characterize using several optical and spectroscopic techniques.
2. The morphology of the particles was determined by optical microscopy (Olympus
BX53 mounted with Orca Flash 4.0 Hamamatsu CCD camera, transmission electron
microscopy (TEM) (FEI Tecnai G2), and scanning electron microscopy (SEM) (Zeiss
EVO 50).
3. The concentration of the Au NPs was estimated using UV−visibleabsorbance
spectroscopy (Shimadzu UV-2600).23
4. The particle zeta potentials were obtained using Malvern zetasizer MS-602.

Experimental setup and Impedance measurement:

1. The experimental setup consists of interdigitated electrodes (IDEs) and LCR meter
(Agilent E4980A, 20 Hz to 2 MHz) interfaced with planar interdigitated platinum
microelectrodes (IDEs) on glass substrates (Micrux, Spain), one of the most widely
used electrode layouts in biosensing and electrochemical measurement systems.24
2. Each microelectrode chip was comprised of 40 symmetrical finger electrodes with 100
μm electrode width and 100 μm electrode gap.
3. A small PDMS film of thickness 1.5mm is punched with a 5mm diameter hole
which can hold 20 μL of sample.
4. This PDMS film was kept on the electrode and 10 μL of sample was loaded after
gentle homogenization by hand and the top was sealed by a glass coverslip via
vaccum grease to avoid evaporation.
5. An AC electric field of 100mV and at 1 MHz was applied and data was collected
every 2.5 min for 1 hour.
 6. Each experiment was repeated at least thrice and results were reported as the mean
± SD.
7. To avoid any leakage the PDMS chip was washed with IPA and dried using
Nitrogen gas after every reading and electrodes were also washed with acetone and
DI water after every reading.
8. As the phase angle between the real and imaginary parts was found to be <3° in all
of the measurements (i.e., mostly resistive behavior), the results were reported in
terms of differential impedance ΔZ, calculated by subtracting the signal from buffer
alone from the total signal.

Figure: Schematic of the experimental setup used in our experiments for measuring the
impedance of the suspension. Particle suspension is placed in 5 mm dia PDMS chip.

 Our system consist of PS particles re-suspended in 40 mM HEPES, PIPES, POPSO,

and EPPS zwitterionic buffers of pH 8.5, 8.44, 10.84, and 8.44 respectively to which
an AC electric field was applied of strength 100 mV for the measurement of impedance
response.
 All the experiments are performed at room temperature.
 For the choice of working frequency we have used these zwitterionic buffers to measure
the impedance responses by sweeping frequencies from 1 kHz to 1 MHz.
Effect of HEPES
 Impedance spectroscopy was used to probe the AC conductivity of extremely dilute
colloidal suspensions (2.5 ×10−5 ≤ Φw/v ≤ 4.0 × 10−2) comprising of polystyrene
microspheres (PS; κa ≫ 1 and ζ = −65 mV), gold nanoparticles (AuNPs; κa > 1 and ζ
= −26 mV), and Au-coated PS metallodielectric particles (Au-PS) in HEPES buffer.
 When AC electric fields of strength 10 mV and 1 MHz were applied via 100 μm gap
interdigitated microelectrodes across 10 μL samples, a highly resistive (θcapacitive <
1°) and nonmonotonic response was obtained with particle concentrations at steady
state.
 While the suspensions were less resistive (than the buffer) below a critical
concentration, they became more resistive above it.
 More interestingly, particle−particle interactions took place in suspensions with
concentrations as low as 0.005% w/v.
 We believe this unique behavior is linked to the ion size asymmetry in the HEPES
molecule that provides an ideal microenvironment for counterionic polarization around
the particles.
 The exact mechanism of polarization in HEPES, however, still remains elusive as the
current theoretical models for simple electrolytes fail to explain our data.

Points
 For particle concentration Φ, at as small as 1% volume fraction prevents the use of
dilute suspension models.13
 Cellmodel theories of electrokinetics for concentrated suspensions13,14 were applied
to suspensions of PS spheres at volume fractions between 1% and 18%.15,16
 Experimental permittivity and conductivity spectra for 200 nm diameter particles
suspended in 0.01 and 0.1 mM KCl showed significant differences from the infinite
dilution theory for all volume fractions, while the spectra for suspensions in 1 mM KCl
agreed with the infinite dilution calculation.16
 The standard system of hard charged particles and a simple electrolyte (e.g., KCl).
 Here, we have investigated the effect of a zwitterionic electrolyte, HEPES, on the
dielectric spectra of hard PS and Au particles.
 for the case of colloidal suspensions in a zwitterionic electrolyte wherein the
zwitterionic groups are not localized at the “solid” (lipid)−water interface as in the case
of phospholipid vesicles.
 However, no theoretical models exist at present for predicting the dielectric response
of such systems, which are of interest from both a fundamental point of view as well as
for applications relating to the design of highly sensitive and miniaturized sensors.21
 Knowledge of dependence of electrokinetic behavior of these systems on parameters
such as particle characteristics, surface charge, and particle concentration will
ultimately aid in application of dielectric spectroscopy in these areas.
 To this end, we have used electrical impedance spectroscopy to systematically probe
the conductivity of colloidal suspensions comprising of dielectric PS microspheres (κa
≫ 1 and ζ = −65 mV), metal Au NPs (κa > 1 and ζ = −26 mV), and metallodielectric
Au-PS particle composites in HEPES buffer in the ultralow volume fraction range of 0.0025% ≤ Φ

w/v ≤ 4%.
 RESULTS AND DISCUSSIONS
Deciding suitable medium
The first set of experiments was performed to identify a suitable medium for carrying out these
measurements to obtain the highest sensitivity and lowest limit of detection of the particles.
For this, time-dependent impedance spectra were recorded with three different media, ultrapure
DI water, PBS, and HEPES by sweeping the frequency across the 1 kHz to 1 MHz range. In
all three cases, while the signals decreased rapidly with frequency especially in case of
ultrapure DI water and PBS (a), there was no significant variation with time, suggesting that
the signals were stable.(b)