Biosensors Basics Recognition Transduction

group
Giuseppe Maruccio
giuseppe.maruccio@unisalento.it
Giuseppe Maruccio
Omnics Research Group
Dip. di Matematica e Fisica, Università del Salento,
CNR NANOTEC - Institute of Nanotechnology
Via per Arnesano, 73100, Lecce (Italy)
Biosensors & Lab on chip

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group
Giuseppe Maruccio
3
PARTE VII. Biosensors,
Microfluidics and Lab-on-Chip
 (a) Biosensors. Basics, structure and figures of merit. Recognition elements.
Transduction Methods: calorimetric, mechanical/mass,
electrochemical/impedance, ISFET, Neuroelectronics & Optogenetics, Optical,
Microscopes, Magnetoresistive.
 (b) Microfluidics and Lab-on-chip: Basics and theory, Components and
applications, Lab-On-Chip, Recent Literature overview and new trends: research
in Omnics group, organ on chip, liquid biopsy, breath analysis and wearable
technologies.

group
Giuseppe Maruccio
Definition, components, classification,
glossary, history, Performance factors
& Desirable requirements, etc
Biosensors

VIIa. Nanobiotechnologies  Biosensors
└ Basics └ Recognition └ Transduction └ Temp └ Mass └ EC └ ISFET └ Neur └ Optical └ XMR
 Physical sensors: measuring physical quantities for their own sake
 Chemical sensors: responsing to a particular analyte in a selective way through a
chemical reaction and can be used for qualitative/quantitative determination of the analyte
[R.W. Catterall]
- Biosensors: devices for the detection of an analyte that combines a biological component
(sensing element) with a physicochemical detector component (transducer). [International
Union of Pure and Applied Chemistry]
- Biosensors2: devices that use specific biochemical reactions mediated by isolated enzymes,
immunosystems, tissues, organelles or whole cells to detect chemical compounds usually by
electrical, thermal or optical signals. [IUPAC Gold Book]
Name from the nature of the sensing element, not
from the analyte
Function: Qualitative or quantitative determination
of a particular analyte in a selective way through a
biochemical reaction.
5
Definitions

History of Biosensors
6
YEAR MILESTONE
1930 pH glass electrode
1950 Ames test for urinary glucose
1962 Blood pO2 sensor
1964 Quartz crystal sensor
Valinomycin for ion-selective effect
In vivo Fiber optics Oximeter
1966 Glucose sensor
1968 Fluorescent oxygen sensor
1970 ISFET
1975 First microelectrode with 1 μm
First of many biosensor-based laboratory analysers
Fibre-optic sensor to measure carbon dioxide or oxygen
(and then alcohol by immobilising alcohol oxidase)
1976 Lactate Analyser LA 640: soluble mediator
hexacyanoferrate used to shuttle electrons from lactate
dehydrogenase to an electrode.
1977 Enzyme FET
1980 Fiber optic pH probe
1982 in vivo application of glucose biosensors: first needle-type
enzyme electrode for subcutaneous implantation
1983 First international meeting on chemical sensors
1990 First world congress on Biosensors, Singapore

Integration and wearable sensors
Lab on a chip
7
Wearable sensors

Market
Areas of application
• Health care – measurements of blood,
other biological fluids, ions, metabolites
to show a patient
metabolic state
• Industrial processes, e.g.
Fermentation
• Warfare detection
• Environmental monitoring
• Food control
Common assays in medicine
8
11.6 Billion $ at 2012
%
year

 DYNAMIC RANGE: value of measurand over which sensor is
intended to measure.
 LINEARITY: closeness between calibration curve and straight
line within the dynamic range (maximum deviation expressed in
percentage).
 OFFSET: output with zero measurand applied.
 SENSITIVITY: value of response per substrate concentration:
slope of calibration curve.
 SELECTIVITY: ability to discriminate between different
substances.
OVERLOAD CHARACTERISTICS
 Overload: maximum magnitude applied without causing a
change in performance beyond specified tolerance.
 Recovery time: time after removal of an overload condition
before the sensor again performs within the specific
tolerance.
 DETECTION LIMIT: lowest detectable value, usually
defined as the lowest limit of linear range.
Wide measurement range
(susceptible of saturation?)
9
Output-measurand relationship

Accuracy: agreement between
measured value and actual value.
Precision: extent of random
error in the measurement.
10
Accuracy & Precision

 Repeatability: ability to reproduce output readings repeating the exact measurement at exactly the same condition
within short time period.
 Reproducibility: same after longer period of time or at a different conditions.
11
 Hysteresis: maximum difference in output, at any measurand value, when the value
is approached first with an increasing and then decreasing measurand.
 Reversibility: Continuous measurements (no consumption of the analyte)
Repeatability/Reproducibility & Hysteresis
RESPONSE TIME: Time
necessary for having 95% of
final value in response to a step
change in the measurand
response. A short response time
is derirable for fast
measurements and high sample
throughput.
WORKING LIFETIME
(and shelf time as well):
minimum time over which
sensor will operate without
changing performance
characteristics beyond specified
tolerance.
– Degradation during continuous
use
– Storage in wet condition
– Shelf life (in dry, in original
packaging)
STABILITY: sensor ability
to maintain its performance
characteristics for a time period.

12
Gas sensors
Molecular
recognition
Specificity/Selectivity

based on
Transduction mechanism:
Optical (light/matter interactions,
light emission & luminescence,
SPR), electrical/electrochemical
(redox reactions or electrical
signals), mechanical
(gravimetric/mass, piezoelectric
and cantilever), calorimetric,
magnetoresistive transducers
Placement: In-vivo, in-vitro, point-of-care
13
Recognition Element: Enzyme (e.g.
glucose oxidase), DNA/RNA (gene),
antibodies, receptor protein, whole-cell,
tissue slice (liver, heart).
Classification of Biosensors

Catalytic
Biocatalytic sensors
(Enzyme-based
Detection)
SENSING STRATEGY REACTION
(Catalytic) Enzyme sensors based on substrate
consumption and product formation
(Non Catalytic) Bioaffinity sensors based on capture
Insoluble salt-based sensors
Non-catalytic
Biocomplexing or
bioaffinity sensors
(Antibody-based
Detection)
14
Principal Biosensing Strategies
1. Catalytic and non catalytic strategies
3. Direct, competitive and binding inhibition assays
DISADVANTAGES OF “LABEL-
BASED ” TECHNIQUES
 extra-time and cost
 Labeling can interfere with
molecular interaction occluding
binding sites (false negatives)
 background binding can interfere
(false positive)
 sometimes do not allow
observation of binding kinetics
2. Label-based vs Label-free

Principal Biosensing Strategies
 Various measurement formats to ensure that binding event produces measurable sensor response.
 Most frequently used are:
 In direct detection format, analyte in a sample
interacts with a biomolecular recognition element
(antibody) immobilized on the sensor surface, Fig. 6.
The resulting refractive index change is directly
proportional to the concentration of analyte.
INHIBITION ASSAY
DIRECT
DETECTION
SANDWICH ASSAY
15

16
└ Basics
Biosensors

group
Giuseppe Maruccio
target
The sensing element

group
Giuseppe Maruccio
450.0 500.0 550.0 600.0 650.0 700.0 750.0 800.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Abs
wavelength (nm)
B
100µm
-30 -20 -10 0 10 20 30
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
GMR(%)
Magnetic field (Oe)
empty sensor
with MNPs
Py 5 nm
Ta 10
Co 3
Co 0.5
Cu 2.1
Ta 2
Py 2.5
AlOx 25
0.0 300.0M 600.0M 900.0M 1.2G 1.5G 1.8G
-45
-40
-35
-30
-25
-20
-15
-10
Transmitted
signal
(dBm)
Frequency (Hz)
m=0.9
1st
3rd
5th 7th
9th
11th
13th
The transducer / sensor

Recognition elements
19

1 .Deliver or synthesize the bioreceptors
3. Passivate the surface
4. Stock in appropriate conditions
2. Immobilize bioreceptors
 Self-assembly of thiols on gold films
A. IBM and Agilent Technologies
Inkjet printing of single
stranded DNA
B. Photolithography patterning of
DNA features Affymetrix,
GeneChip® Santa Clara
(California)
(A) (B)
20
Development steps of the sensing surface

Self-assembly

Antigens-Antibodies

DNA origami

Platform for linking biomolecules either using direct chemical linkages
or by encapsulation with the help of polymeric supports.
1. Immobilized enzymes are very much sensitive towards changes in pH, ionic strength and temperature: a
minor change in one of these parameters can sometimes be responsible to loose the biological activity.
2. The chemical stability of some of the SAMs is not very good as monolayer can be chemically oxidized
during the course of investigations.
3. Electric field induced and thermal desorption of monolayers is detrimental to biosensor applications.
4. Due to high surface energy, hydrophobic SAM surface can accumulate several contaminants and
hence unwanted impurities can adsorb and block the analyte recognition sites.
1. Easy formations of ordered, pinhole free and stable monolayers.
2. Membrane-like microenvironment (cellular) suitable for biomolecule immobilization.
3. Flexibility to design the head group of SAM with various functional groups in order to accomplish
hydrophobic or hydrophilic surface as per the requirement.
4. Only minimum amount of biomolecule (monolayer) is needed for immobilization on SAM.
5. Reasonable stability for extended period, allowing several reliable measurements.
6. Ability to unravel molecular level information about phenomena such as protein adsorption, DNA
hybridization, antigen–antibody interaction etc. using surface sensitive techniques such as AFM.
Advantages
Disadvantages
28
SAMs for biosensors

Functional groups
 Interactions strengths highly dependent upon their chemical
natures, ranging from very weak (e.g., n-alkanes adsorbed on
gold or graphite) to strong enough to break chemical bonds
within the molecule (e.g., ethylene on platinum).
 Remarkable SPONTANEOUS SELF-ORGANIZATION into
well-ordered arrays (often both short- and long-range order).
 VARIOUS FUNCTIONAL GROUPS that have been
incorporated into thiol-based SAMs, whether within the interior
(I) of the film or at the terminus (T). Substituents have been
omitted for clarity, and sites of attachment to the remainder(s) of
the thiol-based molecule are indicated by a –’ off of the listed
moiety.
 The incorporation of FUNCTIONAL MOIETIES such as
chromophores, electroactive groups, or molecules that can bond
within the SAM (i.e., covalent cross-linking between adjacent
molecules or non-covalent hydrogen bonding) enable
capabilities in sensing, electron transfer, molecular recognition,
and other areas.
29
Progress in Surface Science 75 (2004) 1–68

 Many technologically relevant materials possess well-defined surface chemistries, including metals,
semiconductors, oxides, and other complex materials such as superconductors
 a variety of heteroatom containing molecules have been shown to self-assemble on such substrates.
Strategies - Functional groups
30

Thiol-based SAMs are attractive
structures for several reasons.
Well-ordered SAMs can be
formed from a variety of sulfur
containing species (i.e., thiols,
sulfides, disulfides [3]), molecules
are stable once adsorbed on the
surface
n-dodecanethiolate monolayer self-assembled
on an atomically flat gold substrate
Alkanethiolates on noble
metal surfaces
The structure as well as the placement of
alkanethiolate films on surfaces other than gold has
been studied and reported, including platinum
[82,132], palladium [105,133], silver [111,134], and
copper [111,135].
31
The n-alkanethiolate SAM

Schematic of an organosilane self-assembled on a SiO2
substrate. In order to form a complete monolayer, the
silane groups condense with surface hydroxyl groups to
form a thin layer of polysiloxane.
• Organosilanes generally consist of a silicon atom
tetrahedrally bound to three similar functional
groups (typically short chain alkoxy groups or
chlorines), and then to a functional group of
interest.
• While the S–Au bond is mostly charge transfer in
nature, the ‘‘headgroup–substrate’’ interaction of
the silane and SiO2 surface is quite different;
here, the silane molecules condense with native
hydroxyl groups adorning the SiO2 surface,
forming a thin layer of covalently linked
polysiloxane at the interface.
• This small polymer interface is much more
thermally stable than its RS–Au counterpart, thus
affording greater stability and an extremely
robust system.
32
Organosilane SAMs on SiO2

Formation of multicomponent SAMs
 SPONTANEOUS PHASE SEPARATION.
 Two differing adsorbates mixed in solution  both species will adsorb.
 If species differ enough in molecular composition, they will aggregate into homogeneous domains to maximize self–self
interactions through van der Waals, hydrogen bonding, or other interactions. n-decanethiol and n-dodecanethiol do not phase
separate if coadsorbed from solution at room temperature but can be separated into homogeneous domains by post-adsorption
techniques such as thermal processing and backfilling;
 The relative fractional surface coverages will not necessarily be that of the coadsorption solution;
 ‘‘HOST–GUEST’’ SELF-ASSEMBLED MONOLAYERS.
 Molecules placed at low fractional surface coverage within a pre-existing SAM matrix.
 incoming (‘‘guest’’) molecules will insert into a (‘‘host’’) SAM at its local defect sites.
 ELECTROCHEMICAL MANIPULATION OF ADSORBED THIOLATES.
 Two or more substantially different adsorbates in solution  phase separation to maximize self interactions.
 Electrochemistry then to manipulate the adsorbates themselves e.g. by electrolytically cleaving Au–SR bond.
 Thiols typically displaced from metal electrodes by applying reductive potentials, yet it is also possible to strip them from
surfaces using oxidative potentials.
 Different thiols have different reductive potentials, varying from -0.75 V (for short mercaptoalkanoic acids) to -1.12 V (for
n-hexadecanethiol, both peak potentials vs. Ag/AgCl electrodes) [149].
 Therefore, it is possible to desorb individual,
small groups, or domains of thiols selectively
without desorbing others, should two or more
thiols be coadsorbed on a gold surface.
33

34
Techniques for SAM patterning

Enzyme SAM for biosensor application
35

Molecularly imprinted polymers

Sensors 2017, 17(7), 1623; https://doi.org/10.3390/s17071623
Tissue-based sensors
Why cell-based biosensors?
- Known ultrasensitivity of cells:
• Olfactory neurons respond to single odorant molecules
• Retinal neurons triggered by single photons
• T cells triggered by single antigenic peptides
- Cell-based biosensors are based on a primary transducer (the cell) and
secondary transducer (device which converts cellular/biochemical response
into a detectable signal)
- Detection of arbitrary targets: trasnsfected cells with receptors to introduce
responsiveness of e.g. neuronal cells to a chosen compound
37
Cell-based sensors

Methods for SAM
characterization
38

Contact angle goniometry (CAG)
1. Drop of water (other liquid) in contact with surface;
2. Angle between the film and liquid droplet measured.
While CAG cannot determine the exact molecular composition, it
can provide a rough estimate of both film quality and overall
hydrophilic character.
Measured angle reflects DEGREE OF SURFACE ORDER, and
can indicate incorporation of functional groups.
Ex. n-alkanethiolate SAM: contact angle slightly different from
water droplet resting upon close-packed, methyl-terminated
surface versus droplet resting upon disordered and thus exposed
methylene tails and interior of the film.
For more hydrophilic surfaces (i.e., –OH, –COOH, or –CO2CH3-
terminated surfaces), the water droplet will make a smaller contact
angle as the water will more effectively wet the hydrophilic surface.
Conversely, a drop of a neat liquid such as hexadecane against the
surface of an n-alkanethiolate SAM will have a very low contact
angle, as the hydrocarbon liquid will spread across the surface of the
film.
39

Superhydrophobicity
Wenzel state Cassie state
40

Demonstration of the Lotus Effect

Intelligent Textiles

X-ray photoelectron spectroscopy
XPS probes the CHEMICAL NATURE of the SAM:
1. Incident X-rays bombard the sample
2. Electrons ejected from core shells of atoms within SAM, then collected and dispersed in an
analyzer;
3. By MEASURING THE KINETIC ENERGIES of the electrons entering the analyzer, the binding
energies are calculated.
4. These are specific to each element and give indications of the oxidation states of the elements as
well.
XPS showed that a covalent bond exists between the sulfur headgroup and the gold substrate, defined
the chemical species and oxidation states of constituent atoms in the SAM, and demonstrated that the
film is of single monolayer thickness [17,41,108,111,137,138].
As it is capable of identifying the elements present and their oxidation states within the SAM, it has
been utilized as a powerful diagnostic tool to analyze SAMs once they have been chemically modified.
Through angular dependent sputtering experiments, in which the X-rays are focused to destroy through
the SAM down to the substrate beneath, the thickness of the SAM can be calculated based on ratios of
the substrate signal before and after the presence of the SAM.
44

Fourier transfer infrared spectroscopy (FT-IR)
FT-IR to measure the vibrational frequencies of bonds within molecules. The vibrational modes of
molecules attached to surfaces can also be probed.
However, specific surface selection rules exist, and can be used to advantage. Only molecules whose
vibrations are perpendicular to the surface will be detected, as the oscillations running parallel to the
surface are effectively cancelled out by the dipole symmetry between the molecules in the film and the
metallic substrate.
The alkyl tails vibrate at characteristic frequencies (in the region of 2800–3000 cm1 [22]); both the
breadth of these peaks as well as the frequencies of the vibrations themselves yield a picture of the
relative order and fraction of chain defects within the SAM. Should the chains be disordered, the
vibrational spectra will show a breadth of the peaks (as the lack of a constraining environment imposed
by neighboring alkyl chains will allow a distribution of vibrational frequencies), as well as a slight
redshift in vibrational frequency.
FT-IR can also identify the presence of particular functional groups by identifying their characteristic
vibrational frequencies (i.e., carboxylates, amides, hydroxyls, etc.). Thus, the progress of reactions at
SAM surfaces can be followed using FT-IR.
RAMAN SPECTROSCOPY has also been used to study SAMs [140,141]; it provides important
information about adsorbate orientation through measured vibrations in the m(C–S), m(C–C), m(C–H),
and m(S–H) regions, which are often too weak to be detected in IR spectra.
45

 Gold substrate as one large electrode
 SAM-modified gold electrode generally placed into an
electrochemical cell with counter- and reference
electrodes.
 Close-packed SAM  up to 99% electrochemical
blocking effect (EBE). ‘‘Pinhole’’ defects can serve as an
array of ultra-microelectrodes.
 To measure EBE, a freely diffusing species capable of
forming a reduction–oxidation pair with the gold surface
is placed in the cell along with an electrolyte; common
choices include
 The metal ions can only be reduced or oxidized at the
gold surface through pinholes and other defects in the
monolayer film, where the species can penetrate through
the SAM and access the electrode underneath [143]; such
pinholes have been shown to be permanently passivated
by the electrochemical polymerization of phenol [145].
46
Electrochemistry

For electron transfer studies through the film, the redox couple is often covalently linked to the monolayer itself. Example:
 probes such as ferrocene (Fc) and pentaaminepyridine ruthenium at the termini of the adsorbates (i.e. at the external
SAM interface).
 alkyl matrix underneath the probes in order to form a close-packed SAM that spatially separates them from the
surface (this alkyl matrix has been manipulated to contain other functional groups in order to probe the individual
contributions of the methylene matrix or other groups to the electron transfer through the film).
 electrons can be drawn from an immobilized Fc down to the gold electrode with positively applied potentials [113].
The rate at which these electrons travel can be measured, and the rates at which the electrons move through the film
are materialdependent.
 by controlling the spatial distribution of electroactive adsorbates (i.e., alkanethiolates terminated with Fc) diluted
within methyl-terminated alkanethiolates, highly sensitive ultra-microelectrode arrays can be incorporated into a
SAM. The presence of conjugated groups in the interior of the film (such as phenylene–vinylene, phenylene–
ethynylene, alkene, or alkyne moieties) can greatly accelerate the electron transfer through the film, as electrons can
move through delocalized pathways as opposed to r pathways in alkyl networks [25,49].
 As electrons can be moved controllably through a SAM, electrochemistry can also be used to reduce or to oxidize
pendant groups at the solution–film interface that may be poised for further reaction [65,146,147], as well as to
reduce or to oxidize the headgroup so as to desorb the molecule in order to perform post-adsorption processing on the
SAM [148–150].
Electrochemistry
47

Phase Imaging
with chemical sensitivity
LFM
Scanning probe microscopes
EFM
48

Calorimetric/
Thermometric
sensors
49

 Measurement of heat generated by an enzymatic reaction (enthalpy change).
 Enzyme provides selectivity and reaction enthalpy cannot be confused with other reactions.
Enzyme reaction
Catalytic gas sensor
Thermal conductivity devices
(typically gas chromatography)
 Ideally total heat evolution by a calorimetric measurement but always heat loss in real systems (not adiabatic
process)  temperature difference before and after evolution measured most often.
 heat capacity of specimen and container assumed constant over small temperature range measured.
 Simplest transducer: thermometer coated with the enzyme.
 Thermistors typically utilized to transform heat into electrical signal (change of resistance with temperature).
Note. The change of resistance of certain oxides is much greater than the change of length of a mercury column or the
microvolt changes of thermocouple junctions.
50
Calorimetric readouts

Measurement of reversible reactions among biomolecules  determination of binding constants, reaction
stoichiometry and the thermodynamic profile (enthalpy and entropy) of the interaction in a single experiment
without immobilization/modification of reactants and/or labels.
• Two cells kept at the same temperature: heat sensor detects temperature difference and feedback to the heaters which
compensate to return the cells at equal temperature
• ligand solution titrated into a well-insulated, stirred cuvette containing a receptor kept at constant temperature.
• As heat is released or absorbed during a molecular interaction, a BINDING ISOTHERM is obtained as a plot of the
heat change versus the molar ratio of ligand to receptor.
• control experiments to compensate for bulk effects such as heat of dilution of ligand and receptor and heat of mixing
51
Isothermal titration calorimetry (ITC)

Isothermal titration calorimetry (ITC)
Malvern video
Measurement of reversible reactions among biomolecules  determination of binding constants, reaction
stoichiometry and the thermodynamic profile (enthalpy and entropy) of the interaction in a single experiment
without immobilization/modification of reactants and/or labels.
Example: exothermic reaction
• Two cells kept at the same temperature: heat sensor detects temperature difference and feedback to the heaters which
compensate to return the cells at equal temperature
• ligand solution titrated into a well-insulated, stirred cuvette containing a receptor kept at constant temperature.
• As heat is released or absorbed during a molecular interaction, a BINDING ISOTHERM is obtained as a plot of the
heat change versus the molar ratio of ligand to receptor.
• control experiments to compensate for bulk effects such as heat of dilution of ligand and receptor and heat of mixing

 DSC measures conformational changes in macromolecules and has been used in the elucidation of thermally induced
structural transitions, estimation of formulation stability, and chemical half-life studies.
 Whereas in ITC assays, Tsample is kept constant, in a DSC assay differences in heat generated in a sample and
reference cell are measured as Tsample is either increased or decreased.
 Heat difference between sample and reference is related to the conformational energy of the receptor–ligand.
 Determination of temperature (TM) and heat capacity (Cp) of transitions very detailed information on :
 dynamic structures of
biological macromolecules.
 interaction reversibility by
cycling the temperature
back and forth.
DSC has been applied
successfully to the study
enzymes, receptors, growth
factors, cell adhesion
molecules and other targets.
Differential scanning calorimetry (DSC)

 QCM & BAW (Bulk Acoustic Wave), SAW (Surface
Acoustic Wave) : they are sensors of Mass and Pressure.
 MicroCantilever : they are sensors of Mass and Surface
Stress
Mechanical/Mass
sensors
54

QCM, BAW, SAW sensors
55

Piezoelectric materials and their applications
The Piezoelectric effect:
Piezoelectric
ceramics for
hi-resolution
(<nm) mid-
range (100
mm) actuators
Inverse-Piezoelect. Effect
Applied force  surface electrical charge

Quartz Crystal – Crystalline SiO2
Only specific materials, belonging to certain
crystal classes, show the piezoelectric effect:
quartz, cadmium sulphide, lithium niobate, lithium
tantalate, zinc oxide, lead zirconium titanate
(PZT), III-nitrides group (GaN, AlGaN).
Piezoelectric effect
57

Quartz Crystal – cut & AW generation
Acoustic wave generation
1. Applied Vac  time-varying mechanical displacement along field direction 
generated acoustic wave propagates until reaching a boundary where is reflected.
2. Piezoelectricity  reflected wave generates charge separation  time-varying
electric field in phase with mechanical displacement  electric potential back in
the electrode regenerating an acoustic wave
RESONANCE if this induced electric field is reapplied to
the device in phase with the displacement  standing wave
58
First piezoresonator employed as a chemical sensor was a AT-quartz resonator
 X plate crystals: large voltage generated when compressed and decrease in frequency
with T increases
 Y plate crystals: large voltage generated by shear stress and increase in frequency
with T increases
 X cuts exhibits an extensional vibration mode with AC voltage
 AT cuts (35 degrees off the Y axis) vibrates in the thickness shear mode
 Two metal electrodes on a thin slice of quartz cut with a specific orientation relative
to the crystallographic axes (AT-cut)
 Displacement in a thickness-shear mode, in which entire material subject to
displacement
AT-cut of quartz crystal
no net gain or loss in energy through the process of
generation, propagation, and regeneration. Due to the
damped wave motion (frictional losses, …), an extra
electric energy has to be supplied to each regenerated wave.

AT-cut of quartz crystal
Quartz Crystal Microbalance (TSM-BAW)
 First piezoresonator employed as a chemical sensor
was a AT-quartz resonator
 Two metal electrodes on a thin slice of quartz cut with
a specific orientation relative to the crystallographic
axes (AT-cut)
 Displacement in a thickness-shear mode, in which
entire material subject to displacement
Resonance condition: thickness equal
to an odd number of a half wavelength
Typical d = 0.2 mm → f0 ~ 9MHz
59

Mass sensitivity of QCM
Sauerbrey (1959) - For small mass changes, the added mass can be treated
as an additional mass of quartz with its corresponding added thickness.
• The change in thickness, Δd, causes a change in the
oscillation frequency, Δf0.
• An increase in thickness and mass produces a decrease in
frequency.
60
𝑀𝑞 = 𝜌𝑞𝑉 = 𝜌𝑞𝐴𝑑
𝑑 =
𝑀𝑞
𝜌𝑞𝐴
∆𝑑 =
∆𝑀𝑞
𝜌𝑞𝐴
&
= −
∆𝑀𝑞
𝜌𝑞𝐴𝑑
= −
∆𝑀𝑞
𝜌𝑞𝐴
2𝑓0
𝑣𝑏
∆𝑓0
𝑓0
= −
∆𝑑
𝑑
= −
∆𝑀𝑞
𝜌𝑞𝐴
𝜌𝑞𝐴
𝑀𝑞
= −
∆𝑀𝑞
𝑀𝑞
Sauerbrey equations describe relationship
between resonant frequency and mass deposited.
∆𝑀𝑞

Initially believed impossible due to excessive wave damping by acoustic coupling into the liquid, but
stable resonance can be obtained if only one face of the QCM resonator is in contact with the liquid.
 Due to the strong adhesive forces between the liquid phase and the surface there is a strong
coupling of the wave motion from the surface into the liquid
 Due to the moderate cohesive forces in the liquid (much smaller than in a solid), the motion
damping is very accentuated.
decay rate of liquid compressional waves and amount of liquid-coupled energy depends on
liquid viscosity. The lost of acoustic energy results in a change in frequency/attenuation
61
QCM - Liquid operation

Quartz plate coated with gold
electrode plus a sensitive
layer (e.g. DNA probes)
Absorption-Based Sensors
63

Cooper et al., 2001

Mechanical shift of resonance for detection of mass change (due to adsorption or chemical reaction)
External parameters changing resonant frequency by modifying
the characteristics of the propagation path:
 mass of landing material (coating or particles) on the surface
 pressure
 temperature
 geometrical properties
Acoustic Wave Sensors
Acoustic waves:
• Bulk Acoustic Wave (through the material)
– TSM (thickness shear mode-QCM),
– SH-APL(Shear-horizontal acoustic plate mode)
• Surface Acoustic Wave (on the surface)
– SH-SAW (Shear-horizontal acoustic wave) or STW
– Vertical SAW
LIQUID ENVIRONMENT (see later)
If amplitude normal to surface, energy radiated
into the liquid can cause excessive dumping and
energy losses. Mode propagation adapted to
liquid environment:
 TSM (BAW)
 SH-APM (BAW)
 SH-SAW (SAW)
Frequency range of generated elastic wave:
from 1MHz to few GHz
65

Surface Acoustic Waves
SAW properties
 Wave bound to the surface  effectively is an evanescent wave
 Small amplitude (~ 10Å) compared with wavelength and falls
off exponentially with distance from surface
 Penetration depth of wave into the substrate varies inversely
proportional with frequency
66



2



wave
acoustic
group
k
v
v
compressional or primary (P) wave
(bulk longitudinal)
shear or secondary (S) wave (bulk transverse) (Surface) Rayleigh wave
+
(Surface) Love wave
 For f0~200MHz, 3 pg of
biomolecules are detectable
 3 order of magnitude more
sensitive than BAW

SAW Excitation and Detection
Elastic and Viscoelastic Films
on a SAW Device
 For f0~200MHz, 3 pg of
biomolecules are detectable
 3 order of magnitude more
sensitive than BAW

Surface Acoustic Waves - Applications

group
Giuseppe Maruccio
SAW is a widely used technology
Quantum
technologies
ICT
Touch screens
Filters
mobile phone Sensors
RFID
Surface acoustic waves

group
Giuseppe Maruccio
MEMs accelerometer
Surface acoustic waves

SAW - Applicazioni

group
Giuseppe Maruccio
0.0 500.0n 1.0µ 1.5µ 2.0µ 2.5µ 3.0µ
-0.04
-0.03
-0.02
-0.01
0.00
0.01
0.02
0.03
0.04
Transmitted
signal
(Volt)
time(s)
d
357.9M 358.1M 358.2M 358.4M
-11
-10
-9
-8
-7
-6
-5
Reflected
signal
(dBm)
Frequency (Hz)
500 mirror lines, 20 emitter IDT
SAW devices in Lecce

group
Giuseppe Maruccio
Applications
• Sensing
SAW Delay lines : High Order Harmonic Mode excitation
S.Rizzato at al., J. Micromechanics and
Microengineering 27, 125002 (2017)

group
Giuseppe Maruccio
SAW Delay lines on Gallium Nitride
R L
PB
Output IDT
Input IDT

group
Giuseppe Maruccio
100 200 300 400 500 600 700 800 900 1000
-110
-100
-90
-80
-70
-60
-50
-40
-30
-20
Amplitude
(dBm)
Frequency (MHz)
190 195 200 205 210 215 220 225
-90
-80
-70
-60
-50
-40
-30
-20
Amplitude
(dBm)
Frequency (MHz)
SAW
PS-BAW
SAW Delay lines on Quartz

group
Giuseppe Maruccio
0 16 32 48 64 80 96 112 128
0.0 2.9 5.8 8.7 11.6 14.6 17.5 20.4 23.3
-50
-40
-30
-20
-10
0
Mass (ng)
(°)
Number of particles (x104)
-30 0 30 60 90 120 150 180 210 240 270 300
-100
-90
-80
-70
-60
-50
-40
-30
-20
-10
0
10
1 mm
200 nm
40 nm

(°)
Mass (ng)
0 16 32 48 64 80 96 112 128
0.0 3.6 7.3 10.9 14.5 18.2 21.8 25.4 29.1
-50
-40
-30
-20
-10
0

(°)
Number of particles(x106)
Mass (ng)
0 40 80 120 160 200 240 280 320
0.0 1.1 2.3 3.4 4.5 5.7 6.8 8.0 9.1
-100
-80
-60
-40
-20
0

(°)
Mass (ng)
Number of particles (x109)
Quartz 200 nm 1 µm
40 nm
SAW-based sensor for the detection of nanoparticles
S.Rizzato at al., J. Micromechanics and Microengineering 27, 125002 (2017)

Cantilever-based sensors
Read-out schemes:
1. Static bending
2. Frequency change
Reference required in both cases
79

produced by microfabrication
using dry- and wet-ething
Read-out strategies
• Mechanical Displacement (Bending)
– induced by surface stress
– detect a single base mismatch between two 12-mer olinucleotides
– analogous to the effect on the lateral tension of a lipid bilayer
produced by the interaction between membrane molecules
Cantilevers
• Change in the resonance frequency
– induced typically by mass changes
– analogous to QCM sensor but more easily miniaturizable and suitable
for high density array (a different probe for each cantilever)
Functionalization

Mechanical Displacement
Asymmetric Surface
Stress between the
functionalized part and the
opposite one
Differential deflection: about 10 nm
for a 16-mer oligonucleotide target.
81
Fritz et al. Science 288, 316 (2000)

Harmonic motion, damped by sample interaction
kz
z
m 



z
m
k
z 



t
sin
A
z 0


test solution:
Cantilever as a spring
m meff
effective mass
E = Young modulus
I = moment of inertia.
spring constant (stiffness) in normal direction
f0
resonance frequency
f0
For rectangular cross section:
Ex. h= 0.5 µm, w= 10 µm, l = 100 µm
E = Young modulus: SiO2 = 0.6 1011 N/m2
 = density : SiO2 = 2.2 103 kg/m3
f0  250 kHz
(typical in air)
82
z
z
2
0





0
0
0
0
f
1
T
,
2
f
,
m
k








Frequency change by added mass
1. Attachment of small mass M to cantilever end
b
a
M
m
m
f
M
m
k
2
1
f
f 0
1
0






2
1
2
0
2
1
f
f
f
M
m


destructive measurement
no end mass
44 ng end mass
2. Resonance frequency shift to calibrate k
)
f
/
1
(
)
f
/
1
(
M
)
2
(
k 2
0
2
1
2



83

Change in resonant frequency
A cantilever array oscillated at its resonance frequency can be used as a mass
detector. Molecules from the environment diffuse into the coating of the
cantilever. The mass change can be determined from the shift in resonance
frequency.
A typical mass change of 1 pg/Hz is observed.
Tipical resonant frequency: few hundreds kHz.
84

Living cantilevers
Silicon cantilever
Park et al, Lab Chip, 2008, 8, 1034–1041

Piezoresistive cantilevers
86

EC sensors
87

Principle of electrochemical Biosensors
History of Biosensors
1956: Clark paper on the oxygen electrode.
1962: "how to make electrochemical sensors
more intelligent" by adding "enzyme transducers
as membrane enclosed sandwiches”. → First
biosensor where an amperometric oxygen
electrode was immobilized with an enzyme
(glucose oxidase) [1].
the blood glucose biosensor
L. C. Clark and C. Lyons, Ann. N.Y. Acad. Sci., 1962, 102, 29–45.
88

Electrochemistry
 deals with electron transfer (redox) reactions from one substance to
another  measurable current
 measurement of electrical quantities such as potential, current, and
charge in order to gain information about composition of solution
and reaction kinetics.
 APPLICATIONS: Batteries, Corrosion, Industrial production of
chemicals , Biological redox reactions, biosensing
89

Electrodes in solutions & EC cell
Piece of metal in solution containing ions
 charge separation across metal-solution boundary
 This sets up a potential depending on:
• Nature of electrodes
• Nature and concentration of solution
• Liquid junction potential (or potential across the
membrane)
Can we find E for half-cell reaction?
Not directly, it requires a second half cell or a
reference electrode.
Electrochemical Cell
• Any redox reaction can be expressed as difference
of two half-reactions: Ox+ν e− →Red
ANODE (CATHODE): electrode
where oxidation (reduction) occurs.
90
OXIDATION: loss of electron(s)
by a species; increase in oxidation
number; increase in oxygen.
REDUCTION: gain of electron(s);
decrease in oxidation number;
increase in hydrogen.
OXIDIZING AGENT: electron
acceptor; the reagent is reduced
REDUCING AGENT: electron
donor; the reagent is oxidized.
half cell
Electrochemical cell
TERMINOLOGY
STANDARD CELL POTENTIAL, E0: quantitative measure of reactants tendency
to proceed to products when all are in their standard states at 25 °C.
For Zn/Cu cell, potential is +1.10 V at 25°C and when [Zn2+] and [Cu2+] = 1.0 M.
emf (electromotive force):
 is the difference between the electrode potentials of the two half cell
 depends on nature of electrodes, nature and concentration of common solution,
liquid junction potential across the salt bridge.

Three-electrode cell & Reference Electrodes
Precise control of the potential at the electrode:
 Working electrode (WE). The most important electrode in CV is the
working electrode. It can be made from a variety of materials including:
platinum, gold, silver, glassy carbon, nickel and palladium. Redox of the
analyte takes place here.
 Counter (Auxiliary) electrode (CE). The auxiliary electrode is also
known as the counter electrode. Its purpose is to conduct electricity from
the signal source into the solution, maintaining the correct current.
 Reference electrode (RE). electrode having reproducible potentials in
solution with low coefficient of variation with temperature. No current
through RE ideally. RE is used to provide precise control of potential at
the WE, and the current from WE to CE is measured.
Many varieties of REs; most common and available commercially:
91
Primary Reference Electrode
• Standard Hydrogen
Electrode (SHE):
[H+]=1M, P(H2)=1
atm, T= 298K
• E=0.000V
• Very reproducible
(±10 μV)
Secondary Reference Electrodes
• Silver-Silver Chloride Electrode (E=0.225V saturated)
• Saturated Calomel Electrode (SCE) (E=+0.242V
saturated)

Nerst Equation
A cell where overall cell reaction has not reached chemical equilibrium can do electrical work as the reaction drives electrons
through an external circuit
we,max=ΔrG=-νFE
where ν = number of electrons transferred
(number of moles of electrons involved),
F=eNA = Faraday constant (96485 C mole-1).
where
aR (aOx) represents chemical activities of
all species on the reduced (oxidized)
side of electrode reaction
T = temperature in Kelvin
R=molar gas constant (8.314 J/K)
Ox + ν e- = R
Special cases
92
In electrochemistry, the Nernst equation relates the reduction potential of an electrochemical reaction to the standard
electrode potential, temperature, and activities of the chemical species undergoing reduction and oxidation. [Wikipedia]
Measured against SHE, [C]=1 M, P=1 atm, T= 25°C

Electrochemical methods/read-out
 Potentiometry: Electrode and solution are in chemical
equilibrium, current flow is near zero, and the the cell
emf (potential) is measured relative to a reference
electrode. The emf is proportional to the logarithm of
the concentration of the substance being determined.
 Voltammetry (Amperometry): increasing (decreasing) potential applied to the cell until oxidation (reduction) of
substance to be analyzed occurs and there is a sharp rise (fall) in the current. Height of peak current is directly
proportional to concentration of electroactive material. If appropriate oxidation (reduction) potential is known, one
may step the potential directly to that value and observe the current.
 Chronoamperometry: concerning monitoring of a RC electrical decay of the cell after an input voltage step.
 Conductometry: Most reactions involve a change in the composition of the solution. This will normally result in a
change in the electrical conductivity of the solution, which can be measured electrically. All ions in solution
contribute to conductance, so it is not specific.
 Coulorometry: analyte specifically and completely converted due to direct or indirect electrolysis, and the quantity of
electricity (total charge in coulombs) consumed in the reaction is measured.
 Impedance Spectroscopy: involving complex impedance measurement with alternating sinusoidal signals
 Field-Effect Device: A transistor is adapted to be a miniature transducer for the detection and measurement of
potentiometric signals, produced by a bio-process on the gate of the device
93
Advantages of electrical output
 Fast measurements
 Sensitive: low detection limits typically ~10-9M
 Electrical signal better suited for signal transmission; multitude of
microelectronic circuits available for amplification, filtering, modulation,
etc.; many options for information display and or recording

Electrochemical Processes
At an electrode surface, two fundamental electrochemical processes can be distinguished:
 CAPACITIVE PROCESS.
 no reaction involved but capacitive currents caused by the (dis-)charge of electrode surface as a result of changes in
area size (dropping mercury electrode), by a potential variation, or by an adsorption process.
 Under potentiostatic conditions, this process tends to be very fast  resulting current usually expires in a few
milliseconds and can thus be reduced by choosing slower scan rates or pulse widths of longer duration.
 NB. In high resistance media, capacitive current will need a substantially longer period to fall off (time constant:
RC, where R = resistance and C= capacitance).
 FARADAIC PROCESSES.
 Faradaic currents IF as a result of electrochemical reactions at the electrode surface.
 Measuring IF  useful parameters as concentration and diffusion coefficient of a species.
 Peak potential (i.e. position)  nature of the species.
 Usually under potentiostatic conditions, faradaic currents are slower to diminish than capacitive currents.
However, when reactant depletion occurs, a faradaic current will also decrease with time. The scan rate/pulse
duration should therefore be chosen slow/long enough to reduce the charging current, without letting the magnitude
of the faradaic current decline below noise level.
 To study electrode kinetics another strategy must be followed. Due to limiting effect of mass transfer, influence of
reaction rate, or corrosion resistance only expressed on short time scales, making necessary to employ short pulses,
fast scan rates, or high frequencies. In such cases, there might be an overlap with capacitive current. Usually in
these cases, one will employ a range of scan rates, pulse duration’s, or frequencies allowing for a detailed analysis
of the electrochemical components and their impedance’s.
94

Production of pH electrodes

Cyclic voltammetry
 Probably most popular EC technique for solid electrodes. CV traces electron transfer during a redox reaction.
SOME APPLICATIONS:
 Probe coupled chemical reactions: particularly to determine
mechanisms and rates of oxidation/reduction reactions.
 Study electrode surfaces.
ADVANTAGES
 reproducible results (invaluable for relatively badly defined
electrode surfaces).
 reduction/oxidation waves observable simultaneously (helpful
in the investigation of electrode processes).
 Several electrode kinetic and electrosorption processes can be
studied in detail from the analysis of cyclic voltammograms
recorded at various scan rates.
Analyte &
Electrolyte
Reference
Electrode
Auxiliary
Electrode
Working
Electrode
1st cycle 2nd cycle 3rd cycle
Time (seconds)
Potential
Initial potential
Final potential
Term Definition
Switching
potentials
initial and final potentials of working electrode
(cycled potential = VWE-VRE).
Epc ; ipc cathodic peak potential; highest current value
Epa ; ipa anodic peak potential; lowest current value
 Electrodes in cell containing analyte (undergoing redox
reaction) and supporting electrolyte.
 Cell also contains two tubes connected to N2 tank used to
purge out oxygen-containing air from cell.
 Inclusion of backward scan (2 vertex potentials instead of
start- and end-potential).
 Often more cycles repeatedly in sequence as part of
electrode conditioning process, or to monitor EC processes
with time.
IMPLEMENTATION
• The system starts off with an initial potential at
which no redox can take place.
• At a critical potential during the forward scan, the
electroactive species will begin to be reduced.
• After reversal of potential scan direction and
depletion of the oxidized species the reverse
reaction, oxidation, takes place.
96

Analysis of an Example K3Fe(CN)6
 Starting at initial voltage (A), then potential scanned.
 At B, potential negative enough to start cathodic current between
species, reducing the analyte at working electrode.
 Reaction continues until most of the species has been reduced,
peaking the cathodic current at (C).
 Current then decays until potential scan reversed (D).
 Scan in positive direction proceeds similarly: cathodic current
continues to slowly decay until the potential reaches a point to start
oxidation of analyte (E).
 Anodic current then measured as the concentration of the reduced
species is significantly diminished (F).
 Anodic current then decays from this peak.
Case study:
Feasibility study of capacitive
biosensor for direct detection
of DNA hybridization
97

Cyclic voltammetry
Ferrocyanide solution using KCl

Cyclic voltammetry

Anodic Stripping Voltammetry
Two steps:
 during the deposition step: the analyte is electroplated on
the working electrode
 during the stripping step: the analyte is oxidized from the
electrode.
 The current is measured during the stripping step.
 The oxidation of species is registered as a peak in the
current signal at the potential at which the species begins
to be oxidized.
 The stripping step can be
either linear, staircase, squarewave, or pulse.
100
for quantitative determination of specific ionic species.
Example: DP-ASV response of the AG-
NA/Bi nano composite modified GCE

Anodic Stripping Voltammetry

DPAnodic Stripping Voltammetry

Chronoamperometry
• Potential stepped in a square-wave fashion to a potential just past where the peak would appear in linear-
sweep voltammetry.
• The current is then monitored as a function of time.
103

Conductometry
Conductance is the inverse of resistance (unit: Siemens)
• Measure of the ease of passage of the electric current
• Related to the dimension of the cell
• Related to the concentration
• Related to the specific ion
104
Conductance= k A/l,
where k: conductivity (S/cm),
also given in molar conductivity
(S/mol cm)

Impedance
spectroscopy
APPLICATIONS
 Probe linearity of electrical/electrochemical reactions.
 Characterize electrical activity across interfaces to determine carrier concentration, etc.
 Determine diffusion rate of counter ions, e.g. in conducting polymer/CNT composites.
 Determine corrosion rates of materials.
 Measure capacitance/resistance of SAMs to ascertain thickness and packing quality.
105

Impedance in ac circuits
106
V = Z I (generalized Ohm law)
Series
and
Parallel
combination
of
impedances

Impedance Spectroscopy
When E is applied as a sinusoidal
function in a linear system, I response
can be represented by a sum of
sinusoidal functions with phase shifts.
Transfer function can be plotted in Nyquist or Bode form.
Impedance in complex plane
 EC interface deplaced from equilibrium  permanent charge and mass fluxes :
• Electrochemical reaction responsible for charge transfer
• Gradients of electrical and chemical potentials responsible for transport of reacting species
 Primarily due to charge transfer which leads to a faradaic current : IF = I0 exp bV (Tafel law, exponential activation law where b = Tafel
coefficient), the steady-state current-voltage curve of an EC interface is non-linear.
 To analyze this non-linear system, a low amplitude perturbation signal (1-10mV) is employed to remain in the linear approximation
 In usual frequency range (10-3 Hz-50kHz), admissible domain is wider for high than for low frequencies where faradaic processes impose
their non-linearities.
107

Esempi Circuitali
1. Notevole differenza nelle costanti di tempo (1000 volte)
 diagramma di Nyquist:
secondo elemento circuitale
a più alta frequenza visibile
solo con forte
ingrandimento.
 Parte immaginaria del
modulo complesso in
funzione della frequenza 
due evidenti massimi di
risonanza, da cui calcolare le
rispettive capacità parallelo.
due circuiti
2. Differenza minore (10 volte) nelle costanti di tempo 3. Influenza della capacità sulla risposta in frequenza
(a) condensatore in parallelo, due R uguali ed una C variabile, con valori
progressiv.te convergenti (C = 1, 10, 30, 100, 1000 nF);
(b) condensatore in serie (C = 10, 100, 500, 1000 nF), un semicerchio
seguito da un andamento lineare verticale che evidenzia la tipica
risposta di un condensatore puro in serie in serie
108
Funzioni «serie»: impedenza Z e modulo complesso M=jωZ
Funzioni «parallelo»: ammettenza Y=1/Z e permittività ε=Y/jω
con la relazione M=1/ε

Example 1
Example 2
Example 3
Example 4
Esempi Circuitali 2
109

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
ELECTRICAL DOUBLE LAYER
+
+
+
+
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
-
-
-
-
-
- -
-
-
-
-
-
-
-
-
-
-
-
In soluzione
Regardless of mechanism by which surface charge acquired, ions of opposite sign (“counter-ions”) in adjacent solution are
drawn to its vicinity, while ions of like charge (“co-ions”) are expelled.  an electric double layer, consisting of a charged
surface with an adjacent layer of the solution containing a net excess of counter-ions such that the whole structure is
electrically neutral.
Helmholtz model (1879)
Simplest conception of
double layer is a
molecular capacitor in
which a monolayer of
counter-ions sufficient in
amount to effect charge
neutralization, is adsorbed
to the surface.
Guoy-Chapman model (1914)
More realistic: it considers the layer of counter-ions to be diffuse,
striking a balance between the ordering effect of the electric field due
to the surface charge and the disordering effect of thermal motion.
Thermal motion of the
ions renders such a picture
untenable, however.
110

ELECTRICAL DOUBLE LAYER & Z-potential

Equazione
cerchio
Circuito equivalente nel caso in cui il fenomeno
principale sia il trasferimento di carica
Trasferimento di carica e Circuiti equivalenti
elettrolita
interfaccia
elettrodo
di lavoro
Celle elettrochimiche abituali: circuito rappresentabile attraverso resistenza
nell’elettrolita R0 in serie ad una maglia ZC rappresentativa della sola interfaccia
dell’elettrodo di lavoro, stante la particolare scelta del controelettrodo (un metallo nobile).
Se la reazione all’interfaccia è un semplice trasferimento di carica, come accade in molti
sistemi reali, il circuito elettrico è ulteriormente semplificato ed alla impedenza faradica si può
sostituire il simbolo Rct, relativo al semplice trasferimento di carica.
L’impedenza del circuito in questione è data da:
Si tratta dell’eq. di un cerchio di raggio Rct/2 e centro in (R0+Rct/2, 0), con la frequenza al
punto di massimo pari a : fmax = 1/ (2π Rct Cdl)
Diagramma di Nyquist
rappresentativo di un
sistema in cui il
trasferimento di carica è
il principale fenomeno
all’interfaccia.
Metallo coperto da isolante. In presenza di uno strato isolante, ma poroso, come può essere una vernice, il circuito equivalente
può essere rappresentato nel modo seguente:
Rpf rappresenta la resistenza al moto degli ioni offerta dalla
porosità dello strato di vernice e Cpf è la capacità del film di
vernice come dielettrico.
112

Diffusione: Impedenza di Warburg
La possibilità dell’instaurarsi di un processo diffusivo
è modellata con l’inserimento di una impedenza di
Warburg ZW, in serie con la resistenza a trasferimento di
carica Rct.
Circuito equivalente rappresentativo di un metallo
ricoperto da uno strato in materiale isolante in cui
sono importanti fenomeni di diffusione.
Il valore dell’impedenza di Warburg è:
ZW = σ w -1/2 (1-j)
dove σ è il coefficiente di Warburg che può avere un valore
tra 0 e 107.
L’impedenza di Warburg si manifesta alle basse frequenze:
nel diagramma di Nyquist appare come una retta
orientata a 45°.
I valori di σ sono quindi indicativi dell’importanza della
diffusione: maggiore sarà σ, maggiore il contributo
diffusivo ai fenomeni d’interfaccia.
113

Randles equivalent circuit
1947: Randles proposed an equivalent electric circuit for the a cell containing an electrolyte in water solution:
 Double layer capacitance Cdl in parallel with interface charge transfer resistance Rct and Warburg impedance ZW which is related to the
diffusion process of ions towards the electrode. This combination is in series with the resistance of the solution Rs.
Scheme 2. Schematic Faradaic impedance
spectra for modified electrode where :
a) Z is controlled by redox probe
diffusion (low frequencies) and
interfacial electron transfer (high
frequencies).
b) Z is mainly controlled by redox probe
diffusion.
c) Z is controlled by interfacial electron
transfer within entire frequency range.
114
114
Scheme 1. General equivalent circuits for
A) non-Faradaic impedance spectroscopy measurements in the absence of the redox probe.
B) EIS measurements in an EC cell.
C) Same with constant phase element (CPE) depending on roughness of electrode surface.
D) double-layer capacitance, Cdl, that includes Cmod, controlled by the modifier layer.
E) electron transfer resistance, Ret, that includes Rmod, corresponding to the different modifier states.
(A)
(B)
(D)
(C)
(E)

EIS Immunosensors
Interfacial impedance immunosensing: A
bioaffinity interaction between an antibody and
an antigen-functionalized electrode increases
double-charged layer thickness, dl, and inhibits
interfacial EC process of redox probe. Electrode
capacitance, Cdl, and Electron transfer resistance,
Ret, are changed respectively.
Katz and Willner,
Electroanalysis, 15, 913 (2003)
 impedance spectroscopy (EIS), including non-Faradaic impedance measurements resulting
in capacitance sensing, is an attractive EC tool to characterize biomaterial films associated
with electronic elements, thus,allowing transduction of biorecognition events at respective
surfaces.
 different kinds of EIS, such as non-Faradaic capacitance measurements, Faradaic impedance
spectroscopy in presence of an external redox label, in-plane alternative voltage conductivity
measurements, and transconductance measurements with field-effect transistors.
Non-Faradaic and Faradaic impedimetric sensing of
DNP-Ab :
(A) Double-layer capacitance, Cdl for:
a) bare Au electrode.
b) DNP-antigen monolayer electrode.
c) DNP-antigen/DNP-Ab layered assembly.
d) DNP-antigen/DNP-Ab/anti-DNP-Ab-HRP layered
assembly.
e) After biocatalyzed precipitation of insoluble product
7 on electrode.
(B) Faradaic impedance measurements: DNP-antigen
monolayer electrodes treated with
- different concentrations of DNP-Ab for 5 min;
- then anti-DNP-Ab-HRP conjugate for 5 min.
- then allowed to stimulate biocatalyzed precipitation
of insoluble product (7). Impedance spectra
corresponding to
(a) DNP-antigen-functionalized electrode and to
primary interaction of DNP-antigen monolayer
with DNP-Ab at :
(b) 0.5 ng mL-1;
(c) 1 ng mL-1;
(d) 2 ng mL-1;
(e) 8 ng mL-1.
Inset: Calibration plots: Ret at DNP-antigen monolayer-
functionalized electrodes upon the analysis of different
concentrations of the DNP-Ab: a) Ret observed by the
direct interaction of the DNP-Ab with the sensing
interface, b) Ret observed by the amplification of the
initial binding of the DNP-Ab with the biocatalyzed
precipitation of 7. [Fe(CN)6]3-/4-, 1x10-2 M was
employed as redox probe.
115

group
Giuseppe Maruccio
Main Advantages:
 Rapid mesurement
 Label-free
 sensitive
redox couple (10 mM):
K3[Fe(CN)6]/K4[Fe(CN)6] (1:1)
Chiriacò et al., Lab on a chip, 11, 658, (2011)
Microfluidic module with
separate sensing areas,
 20 μL chambers with inlet
and outlet microchannels
for fluid handling
 each one containing an
array of 4 gold IDEs (with
10 μm spacing and width)
EIS Biosensors & biochips

group
Giuseppe Maruccio
Detection of cholera toxin
Chiriacò et al., Lab on a chip, 11, 658, (2011)
Limit of
detection:
10 pM
117
Biochips for environmental threats

group
Giuseppe Maruccio
2008
2010
2013
DNA
Cells
Proteins
Diagnostics
Food
Environment
2016
2018
EIS Biosensors & biochips

group
Giuseppe Maruccio
2018

group
Giuseppe Maruccio
FOOD / ENVIRONMENT
2. Cytotoxicity & Migration assays
Primiceri et al., Biosensors &
Bioelectronics 25, 2711–2716 (2010)
Lab on a chip, 11, 4081, (2011)
M. S. Chiriacò et al.,
Lab on a Chip 13, 730 (2013).
Analyst, 138, 5404 (2013)
MEDICAL
DIAGNOSTICS
1. Immunoassay: PSA & PDAC
M. S. Chiriacò et al.,
Talanta 2015, 142, 57-63.
E. Primiceri et al.,
Analytical Methods, 2016,
8, 3055-3060.
Allergens, toxins,
pathogens, contamination
Multipurpose applications

group
Giuseppe Maruccio
M.S. Chiriacò et al., Scient.
Reports, 8, 7376 (2018)
Appl. 2: Environmental threats - Xylella
Serial dilution of stock 105
CFU/ml solution in PBS.
Spiked samples in healthy
olive leaves homogenate
(serial dilutions obtained from
stock 105 CFU/ml solution).
Calibration curves
Assays on asymptomatic
and infected trees
Sample
dilution
ELIS
A
PCR LOC
1:1 + + +
1:5 + + +
1:10 + + +
1:20 - + +
1:50 - + +
1:75 - + +
1:100 - + -
1:1000 - + -

Compact Voltammetry Cell Kit and Screen-Printed Electrodes

ISFETs
123

Introduction to
semiconductor physics
(Conductors, Insulators, &
Semiconductors)
124

BONDING OF SOLIDS
 Covalent: sharing electrons [poor conductors, e.g., C (diamond)]
 Ionic: exchanging electrons [poor conductors, e.g., salt (NaCl)]
 Dipole-Dipole: weak bonding … molecular solids (e.g., H2O)
 Metallic: sharing electrons by all atoms [good conductors, e.g., Fe, Cu]. Each atom contributes 1 or 2 “free” or
“conduction” electrons that are free to move throughout metal (electron “sea” or “gas”).
Introduction to Solid State Physics
• Conductors: mostly metals
• Insulators: mostly non-metals
Consider two atoms “far” apart ...
 The atoms do not interact, therefore, their orbitals are degenerate.
 As the atoms move closer together and their wavefunctions overlap, the
levels split … each into two levels.
 For three atoms, we get three levels.
MOLECULAR ORBITAL THEORY
125

 For a solid: N (~ 1023) atoms  a quasi-continuum of levels.
 Thus, at the equilibrium position, r0, we have energy bands.
 There can be “forbidden energy zones” called band gaps.
BAND THEORY
Insulator
Band gap
Semiconductor
Conductor
No
gap
CONDUCTOR, INSULATOR, SEMICONDUCTOR
- According to band theory, electrons in a crystal free to move when
excited to unoccupied orbitals of a band.
- In a metal, this requires little energy since unoccupied orbitals lie
just above occupied orbitals of highest energy.
ELECTRON-SEA MODEL: Metal as an array of positive ions
surrounded by delocalized valence electrons.
–Metals are good conductors of electricity because of the mobility of
these delocalized valence electrons.
– A metal also conducts heat well because the mobile electrons can
carry additional kinetic energy.
Introduction to Solid State Physics
126

Magnesium
Sodium
CONDUCTORS are materials in which the highest occupied band is not fully
occupied.
This band is also call the conduction band since in the presence of an external
electric field, the electrons can move to a higher energy state within the band and
thus gain kinetic energy, i.e., a current is produced.
• Example: Lithium (1s2 2s1)
Band Theory - Conductors
• Example: Beryllium (1s2 2s2)
2s shell filled, but 2s and 2p bands overlap producing a conduction band.
Conduction band:
empty 3s
antibonding
Valence band:
full 3s bonding
No gap
Conductor
Conduction band:
empty
Valence band:
full
No gap: conductor
127

Insulators (dielectrics) are materials in which the valence band is fully occupied (at T = 0 K).
Example: Diamond
 Good insulators have band gaps of 5 eV or more. At room temperature, amount of thermal energy (kT )
available to a valence electron is approximately 25 meV. Thus, the probability that an electron will be thermally
excited from the valance band to the conduction band is essentially zero.
 In the presence of an external electric field, the electrons cannot move to a higher energy state and gain kinetic
energy, thus no current is produced.
 If the electric field is large enough, then the field can do enough work to boost a valance electron across the band
gap. Thus, the insulator becomes a conductor. This is called dielectric breakdown.
Band Theory - Insulators
Conductor
No gap
Graphite
Insulator
Band gap = 5.5 eV
≈ 530 kJ/mol
Diamond
Band Gap for Semiconductors
Si 1.1 eV
Ge 0.67 eV
128

MOS devices
129

Doping of semiconductors
p-n junction as a rectifier
Under forward bias, minority carriers diffuse into the
neutral regions where they recombine
130

Enhancement Mode Devices
 VDS → movimento di elettroni tra D e S
 Per VDS piccoli, il canale si comporta come una
resistenza (ID proporz. VDS)
 Maggior VGS → canale/correnti maggiori
Canale ad arricchimento (normalmente chiuso)
MOSFET AD ARRICCHIMENTO
- serve VG per formare il canale (non esiste a VG=0):
 Accumulazione: diodi affacciati, scorre corrente
inversa IS.
 Inversione: VGS inverte localmente il tipo di
portatori nella giunzione MOS → canale
conduttivo tra source e drain (I=V/Rch).
VTH tensione necessaria affinché concentrazione
elettroni mobili sotto l’ossido raggiunga il valore NA
Conduction mechanism
131

MOSFET - Summary
132

ISFET
133

(ISFETS and CHEMFETs: Connecting the ionic to the electronic world)
 struttura di un MOSFET ma metallizzazione di gate sostituita con la serie
elettrolita/elettrodo di riferimento. membrana iono-selettiva (sensibile agli
ioni di interesse) allo scopo di esporre l’isolante alla soluzione elettrolitica
da analizzare.
 La funzionalità dell’ISFET deriva dal fatto che l’isolante è in grado di
generare un potenziale all’interfaccia con la soluzione dipendente dalla
concentrazione di ioni nella soluzione stessa. Come conseguenza, la densità
di carica all’interfaccia e quindi la corrente di drain nel semiconduttore
cambiano in modo misurabile
134
Ion Sensitive Field Effect Transistors
ISOLANTE:
 Sensibilità al pH dalla sensibilità dell’isolante (SiO2, Al2O3, Si3N4, Ta2O5, …)
 Ogni isolante ha una sua struttura superficiale microscopica, con diversi gruppi
in grado di caricarsi se esposti alla soluzione.
 Alcuni gruppi reagiscono con l’analita in modo acido (cioè donano protoni H+)
oppure in modo basico (catturando protoni). → Potenziale superficiale tra i 25
mV/pH del SiO2 e i 59 mV/pH del Ta2O5.
 Tempo di risposta determinato da cinetica reazioni superficiali (di solito ms).
SEMICONDUTTORE: sensibile a Temperatura e Luce  preferibili misure
differenziali usando 2 dispositivi identici affiancati, uno solo dei quali è esposto alle
variazioni di pH.
R.E.: ruolo di fornire una caduta di potenziale costante rispetto alla soluzione. Serve
a polarizzare l’interfaccia e la sua differenza di potenziale rispetto alla soluzione è
stabile e non varia in particolare con il pH.
SiO2
->SiOH surface
is amphoteric thus can
take but can also give
H dependent on pH of
solution.
Thus dependent on
pH we will have a
positive or negative
charge density at the
oxide – liquid
interface.
Reaction with H+
→ a certain amount of
charge will appear at
the SiO2 surface
Chemical reaction
at SiO2 surface

S D
(1) Reference Electrode
(Vref)
VGS VDS
IDS
Silicon Substrate
(2) Solution (Electrolyte)
(3) Membrane (MOSFET gate metal)
(4) Gate Insulator
135
ISFET/CHEMFET sensors
Un ISFET può lavorare a tensioni
costanti (misurando le variazioni di
corrente) oppure a corrente e VDS
costanti, misurando le variazioni di
VGS. Nel secondo caso, le variazioni
di VGS riflettono direttamente le
variazioni di φeo.
Vary the voltage on the electrode that is functioning as a gate in order
to keep the current constant.
Come si polarizza un ISFET

Misure di PH con ISFET
136
ISFET
ADVANTAGES
• No glass thus robust
• Small and highly integratable because compatible with CMOS
• The shape can be adapted for various applications
• Fast response
• Easy manipulation
• Can be stored in dry state
• Easy cleaning (tooth brush + soap!)
• Good sensitivity: 56 - 58 mV/pH
• Good pH range: 2 -12

pH measurements / glass electrodes vs ISFET

Ion Sensitive Field Effect Transistors
138

The use of different type of sensors is recomended to obtain more robust semiconductor-based
chemical sensor arrays
Comparison of Chemical sensors
SEMICONDUCTOR-BASED SENSOR ARRAYS
Potential advantages of integrated circuit technology applied to the field of
physiological data acquisition and water monitoring systems : small size;
reliability; low cost & rapid time response; multi sensor chip; on chip signal
processing
139

140
ISFETs & BioFETs
 potential advantages: small size and weight, fast response, high reliability, low output impedance, on-chip integration of
biosensor arrays and a signal processing scheme with the future prospect of low-cost mass production of portable
microanalysis systems;
 ISFET very sensitive for any kind of electrical interaction at or nearby the gate insulator/electrolyte interface
 ISFET as the key element for BioFETs, even if its ion-sensitive properties are not necessary for the signal transduction
 nearly each (bio-)chemical reaction leading to chemical or electrical changes at this interface can be measured by means of an
ISFET coupled with a bioreceptor.
 basic mechanisms of potential generation for BioFETs are potential changes :
(i) caused by a catalytic reaction product (e.g., between an enzyme and its
substrate);
(ii) caused by surface polarisation effects or the change of dipole moments
(e.g., antigen–antibody affinity reactions or DNA (deoxyribonucleic
acid) hybridisation (under certain conditions detectable by an ISFET);
(iii) coming from living biological systems as a result of more sophisticated
(bio-)chemical processes (e.g., action potential of nerve cells).
Analyst, 2002, 127, 1137
 BioFETs can be classified according to the biorecognition element used
for detection. Based on the hierarchy of biological complexity, they can be
subdivided in: GenFET, ImmunoFET, EnFET, cell-based FET;

Label-free detection of DNA
hybridization
 ssDNA onto the transducer
 complementary DNA added to
the buffered electrolyte.
141
FET basically represents a
surface-charge measuring device
GenFET
ISFET-biosensors
ImmunoFET
 Concept by Schenck (1978).
 Gate modified by immobilising
antibodies or antigens (often in a
membrane).
 antibodies and antigens (or more
generally, proteins) are mostly electrically
charged molecules  formation of
antibody–antigen complex on the gate
leads to a detectable change in the charge
distribution and thus, directly modulates
the drain current.
 theoretically capable of measuring
concentration of immunomolecules with a
very low detection limit and a wide
concentration range of 10-7–10-11 M.
 Ideal conditions that are required in this
coherence are: a truly capacitive interface
at which the immunological binding sites
can be immobilised; a nearly complete
antibody coverage; highly charged
antigens and a very low ionic strength.
Penicillin-sensitive EnFET
EnFET
 Concept by Janata (1976).
 Enzymes as bioreceptors due to specific binding
capabilities and catalytic activity.
 Enzymatic reaction with its substrate can be monitored
by the underlying ISFET.
 Ex. Penicillin-sensitive EnFET: enzyme penicillinase
catalyses hydrolysis of penicillin to penicilloic acid
yielding local pH change near ISFET gate region. Then,
the output signal change will be determined by the
amount of penicillin in the sample solution. (S. Caras
and J. Janata, Anal. Chem., 1980, 52, 1935)
Analyst, 2002, 127, 1137

Enzyme-modified FET (EnFET)
 A wide range of EnFETs reported for the detection of the analytes: glucose, urea, penicillin, ethanol, lactose, sucrose, maltose,
ascorbic acid, lactate, acetylcholine, organophosphorus pesticides, formaldehyde, creatinine, etc. (see Table2)
 With the exception of a few papers on urea-sensitive EnFET based on ammonia- or fluoride-sensitive ISFETs, most reported
EnFETs are built-up of pH-sensitive ISFETs, in which the hydrogen ions are produced or consumed by the enzymatic reaction.
 Most popular enzymes for EnFETs are glucose oxidase,
urease and penicillinase because of their role for detection of
glucose, urea and penicillin in many fields of application.
 The corresponding enzymatic reactions are:
 In practice, for exact measurement of test sample, a pH ISFET/EnFET
differential arrangement is often employed, where the pH ISFET acts
as a reference and is assembled in the same way as the EnFET, but
contains a blank enzyme-free membrane.
 Differential mode measurements have many advantages compared to
single EnFET measurements and allow an automatic compensation of
effects that are caused by some disturbing factors such as pH variation
of the bulk solution, temperature variation, drift of the sensor output
signal with the time, etc.
142
Analyst, 2002, 127, 1137

Electrical detection of single viruses using nanowires
Large surface area to volume ratio  whatever happens at the surface will have a huge
impact on the current through the nanostructure.
Lieber et al., PNAS, 101, 14017 (2004)
Figure. Nanowire-based detection of single viruses. (Left) Schematic shows two nanowire devices, 1 and 2, where the
nanowires are modified with different antibody receptors. Specific binding of a single virus to the receptors on nanowire
2 produces a conductance change (Right) characteristic of the surface charge of the virus only in nanowire 2. When the
virus unbinds from the surface the conductance returns to the baseline value.
143

Electrical detection using nanowires
Large surface area to volume ratio  whatever happens at the surface will have a huge impact on the
current through the nanostructure.
DNA
Proteins
- PSA
Nat.Biotech. 23, 1294
144

NEUROELECTRONICS
145

Action Potentials
Membranes create action potentials by actively changing their permeabilities to ions such as sodium and potassium.
Action potential: term used to denote a temporal phenomenon exhibited by every electrically excitable cell.
 The transmembrane potential difference of most excitable cells usually stays at some negative potential called the
resting potential.
 External current or voltage inputs can cause the potential of the cell to deviate in the positive direction and if the
input is large enough the result is an action potential.
 An action potential is characterized by a depolarization which typically results in an overshoot beyond the 0 mV
level followed by repolarization.
 Some cells may actually hyperpolarize before returning to the resting potential.
Action potentials that
characterize actual tissue have
the roughly rectangular shape
that is suggested by the
preceding calculation, but only
as an approximation.
swing of about 100 mV
between the resting and excited
states (polarized to
depolarized). The change of
permeability from resting to
excited values and then back
again allows the membrane to
generate an action potential.
146

Microelettrodi
• Patch-clamp
• Micro electrode array (MEA)
• Elettroencefalografia (EEG)
• EEG intracranica (iEEG)
Ottica
• Lineare:
- Singolo-fotone
• Non-lineare:
- Due-fotoni
- Generazione Seconda Armonica (SHG)
Elettromagnetica
Magnetoencefalografia (MEG)
Tecniche per la Rivelazione
dell’attività elettrica
147

Patch Clamp Technique
148
The main source of experimental data used to
construct models of cell electrophysiology
[Hamill et al., 1981].
Bert
Sakmann
Erwin Neher
Germany (1991 Nobel Laureates)
Erwin Neher & Bert Sakmann, Premi Nobel per la medicina nel 1991
per lo sviluppo della tecnica del patch-clamp che ha reso possibile la
caratterizzazione di singoli canali ionici

Biosensors Basics Recognition Transduction

Biosensors Basics Recognition Transduction

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