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BIOMEMS

Class IV. Microfabrication of electrochemical sensors


Winter 2011

Dr. Marc Madou


Contents
Ion selective electrodes (ISEs) and CO2 sensor (examples of
potentiometric sensors)
Oxygen sensor (based on the fuel cell principle)
Enzyme based glucose sensor (amperometric) and urea
(potentiometric)
Immunosensor (amperometric)
From ISFET to ISNt FET (potentiometric)
Ion selective electrodes (ISEs)

Ag/AgCl Ag/AgCl

Inner solution

Frit
analyte

Inner solution
Membrane (e.g. potassium,
sodium, pH, etc.)
Inner reference electrode/ inner
solution/membrane/analyte (external
solution)/external
reference electrode
Ion selective electrodes (ISEs)
A traditional pH measurement with a
glass electrode is the best known
potentiometric ion selective electrode
(ISE) (e.g. a thin glass layer with this
composition 22% Na2O, 6% CaO, 72%
SiO2)
There is no change in the inner solution
and there is no actual contact between
inner and outer solution for any
potentiometric probe or sensor
Contact with the solution is always
through the external reference electrode
(Luggin capillary)
Ion selective electrodes (ISEs)

Often reference and glass electrode are


combined in one single structure (How
would you make such a thing ? See
homework Q 1)
The resistance (impedance) of this
sensor is very high (glass layer) so that
the input amplifier of the pH meter must
be very high (the input impedance of
the meter must be at least 100 > than
that of the sensor!)
Very high impedance can make the
measurement noisy. The smaller the
sensor the bigger this problem becomes.
Ion selective electrodes (ISEs)
The so-called Donnan potential is established
on both sides of the glass membrane-the
potential on one side is kept constant through
the internal reference solution while the other
side is determined by the analyte solution
For other ions than protons (cations and
anions) other membranes are available (see
e.g. LaF3 for F- and a wide variety of
polymeric membranes)
Ion selective electrodes (ISEs)
An ion selective polymeric membrane is often
made by mixing an ionophore (e.g.
valinomycin, a natural occuring antibiotic)
with PVC and a plasticizer (to make the rigid
plastic more flexible)
In these types of ISEs one sometimes does not
use an internal reference solution at all or one
incorporates a hydrogel to replace the aqueous
solution . This makes the electrode easier to
handle and store. Especially with no internal
reference electrode drift tends to be larger!
The polymeric ISEs lend themselves well to
miniaturization and cost reduction (it is much
more difficult to miniaturize a glass pH
electrode)
Ion selective electrodes (ISEs)
Evaporated Ag film
By making ISEs planar (e.g.
on a polyimide sheet) many
Insulator layer
sensors can be made in parallel
(i.e. batch fabnrication). From
3D structures to 2D !
Mass production can make
them very small (e.g. 2 by 3
mm), cheap (perhaps Chloridized Ag i.e. AgCl
disposable), reproducible and Hydrogel on reference
electrodes with internal
even electronics might be electrolytes

integrated (see below under


Ion selective membrane
ISFETs)
Carbon dioxide sensor
Gases that react with water freeing or
absorbing a proton in the electrolyte may
be detected by a pH sensitive detector
element e.g. glass or IrOx 1.CO 2 + H2 O
Example gases: CO2, NH3, H2S, etc.

A direct proportionality exists between the
concentration of the neutral gas and the
measured pH e.g. in the case of CO2
( with NaHCO3 for internal electrolyte)
i.e.


aH +

Carbon dioxide sensor

Ecell=Eind Eref (1)

AstheindicatorisonlyH+sensitive,andthepotentialofthereferenceisa
constant(becauseoftheconstantchlorideconcentrationintheelectrolyte),wehave

Ecell


Carbon dioxide sensor (3D)
silverspringcontact

Gaspermeablemembrane
Ir/IrOxelectrode

Ag/AgClelectrode

DuallumenPVCtube
Hydrogel

silverepoxy
epoxy
Carbon dioxide sensor (MEMS version)
A pH, CO2 and oxygen electrochemical
sensor array for in-vivo blood
measurements was made using MEMS
techniques
The pH and CO2 sensors are
potentiometric and the oxygen sensor is
amperometric (see further in this class)
The pH sensor is an ISE based on a pH
sensitive polymer membrane.
The CO2 sensor is based on an IrOx pH
sensor and a Ag/AgCl reference
electrode. .
Electrochemical oxygen sensor (fuel cell)
"Fuel cell" oxygen sensors consist of a diffusion barrier, a sensing
electrode (cathode) made of a noble metal such as gold or silver,
and a working electrode made of a metal such as lead or zinc
immersed in a basic electrolyt (such as a solution of potassium
hydroxide).

Oxygen diffusing into the sensor is reduced to hydroxyl ions at the


cathode:

O2 + 2H2O + 4e- -------- 4 OH-

Hydroxyl ions in turn oxidize the lead (or zinc) anode:

2Pb + 4OH- ------------- 2PbO + 2H2O + 4e-

2Pb + O2 ----------------- 2PbO

Fuel cell oxygen sensors are current generators. The amount of


current generated is proportional to the amount of oxygen
consumed (Faraday's Law).
Enzyme based sensor
Enzymes are high-molecular weight biocatalysts Pt- anode (+)
(proteins) that increase the rate of numerous
reactions critical to life itself
Ag cathode (-)
Enzyme electrodes are devices in which the
analyte is either a substrate (also called reactant)
or a product of the enzyme reaction, detected Immobilized glucose oxidase
potentiometrically or amperometrically (e.g. in cellulose-diacetate with heparin)
Example : glucose sensor substrate (glucose)
diffuses through a membrane to the enzyme Polyurethane membrane
layer where glucose is converted
Both oxygen (which is being consumed) and
H2O2 (which is being produced) can be measured
electrochemically (in an amperometric
technique), or the local pH change can be
monitored (in a potentiometric measurement)
Glucose oxidase (in presence of oxygen)

Glucose H2O2 + gluconic acid


Enzyme based sensor

Amperometric glucose sensor


Anodic
based on peroxide oxidation, +i
l
Plateau of limiting current is i

proportional to the peroxide


concentration which in turn is
proportional to glucose - - - typical
0.6 to 0.8 V vs Ag cathode - +
Glucose oxidase is an oxidase type + 0.6 V
enzyme, urease is a hydrolytic
type enzyme:
-i
Cathodic
Urease
CO(NH 2 )2

Enzyme based sensor

A potentiometric urea sensor may


consist of two pH sensors one with V
the enzyme coated on its surface IrOx IrOx
and one without (the reference
electrode) urease
The electrode with the urease will
sense a local pH change
The pH difference bewteen the two
electrodes is proportional to the urea
concentration
As an example two IrOx electrodes
may be used
Immunosensors
Affinity pairs: An enzyme/ substrate combination is only one example of an
affinity pair, in nature there are many other examples of affinity pairs based on
molecular recognition (think about double stranded DNA)
Affinity pairs exhibit tremendous binding selectivity for each other through
their intricate 3D molecular structures (lock and key)
A much more selective affinity pair than enzyme / substrate pair is the
antigen/antibody pair (AgAb) -- KA (affinity constant) values of 106-1012 LM-1
vs 102-106 LM-1 (as a consequence enzyme sensors may be reversible while
imunosensors are irreversible but much more selective)
In an immunosensor one measures the concentration of either an antibody or an
antigen by measuring an event triggered by the binding of an antigen/antibody-
usually a label is involved (e.g. an enzyme, an isotope, a chromophore, etc.) , a
direct detection of the binding event (without label) is very difficult but is being
attempted in various research labs.
Immunosensors
One example of an immunosensor is an enzyme based immunosensor where the label is an
enzyme--see next slide
Typically an antigen (the same antigen we are trying to determine in the unknown solution)
is labeled with an enzyme (say catalase) and added to the unknow sample in which the
sensor is placed
The labeled antigen competes with native (unlabeled antigen) for reaction with the antibody,
which is immobilized on an electrode surface
Unbound labeled antigen is washed off and substrate for the enzyme (H 2O2 in the case of
catalase) is added
The enzyme decomposes H2O2 and the oxygen is picked up by the underlying oxygen
sensor
The oxygen current decreases with increasing concentration of the nonlabeled native
antigen in the sample solution
The enzyme reaction will produce many detectable species per bound AbAg pair, hence the
name enzyme amplification.
Immunosensors
Enzyme labeled antigen

Immobilized antibody

Antigen Competition for sites on the antibody

Oxygen permeable membrane

Immobilized antibody

Oxygen sensor

Oxygen permeable membrane

Oxygen sensor
Immunosensors

Wash the unbound antigen away and add H 2O2

Oygen is formed

Oxygen permeable membrane

Oxygen sensor

The oygen signal is lower the higher the amount of native antigen
From ISFET to ISNT FET
Homework
1. Design a combination glass electrode. Explain how it
works.
2. Design a planar immunosensor. How could you
incorporate a good reference?
3. Explain how a potentiometric CO2 sensor works.
4. List a list of reasons why the ISFET did not become a
commercial success.