Biosensors and Their Principles

1. Introduction
Biological and biochemical processes have a very important role on
medicine, biology and biotechnology. However, it is very difficult to
convert directly biological data to electrical signal, the biosensors can
convert these signals and the biosensors over this difficulty. In recent
years, thanks to improved techniques and devices, the usage of
these products has increased.
The first biosensor was described in 1962 by Clark and Lyons who
immobilized glucose oxidase (GOD) on an amperometry oxygen
electrode surface semipermeable dialysis membrane in order to
quantify glucose concentration in a sample directly [1, 2]. They
described how "to make electrochemical sensors (pH, polarographic,
potentiometric or conductometric) more intelligent" by adding
"enzyme transducers as membrane enclosed sandwiches".
According to a recently proposed IUPAC definition [3], “A biosensor
is a self-contained integrated device which is capable of providing
specific quantitative or semi-quantitative analytical information using
a biological recognition element (biochemical receptor) which is in
direct spatial contact with a transducer element. A biosensor should
be clearly distinguished from a bioanalytical system, which requires
additional processing steps, such as reagent addition. Furthermore,
a biosensor should be distinguished from a bio probe which is either
disposable after one measurement, i.e. single use, or unable to
continuously monitor the analyte concentration”.
A biosensor is a device composed of two elements:
1. A bioreceptor that is an immobilized sensitive biological element
(e.g. enzyme, DNA probe, antibody) recognizing the analyte (e.g.
enzyme substrate, complementary DNA, antigen). Although
antibodies and oligonucleotides are widely employed, enzymes
are by far the most commonly used bio sensing elements in
biosensors.
2. A transducer is used to convert (bio)chemical signal resulting
from the interaction of the analyte with the bioreceptor into an
electronic one. The intensity of generated signal is directly or
inversely proportional to the analyte concentration.
Electrochemical transducers are often used to develop
biosensors. These systems offer some advantages such as low
cost, simple design or small dimensions. Biosensors can also be
based on gravimetric, calorimetric or optical detection [1].
Biosensors are categorized according to the basic principles of signal
transduction and biorecognition elements. According to the
transducing elements, biosensors can be classified as
electrochemical, optical, piezoelectric, and thermal sensors [3].
Electrochemical biosensors are also classified as potentiometric,
amperometry and conductometric sensors.
The application of biosensor areas [4] are clinic, diagnostic, medical
applications, process control, bioreactors, quality control, agriculture
and veterinary medicine, bacterial and viral diagnostic, drag
production, control of industrial waste water, mining, military defense
industry [5], etc. A few advantages of biosensors are listed below:
1. They can measure nonpolar molecules that do not respond to
most measurement devices
2. Biosensors are specific due to the immobilized system used in
them
3. Rapid and continuous control is possible with biosensors
4. Response time is short (typically less than a minute) and
5. Practical
There are also some disadvantages of biosensors:
1. Heat sterilization is not possible because of denaturization of
biological material,
2. Stability of biological material (such as enzyme, cell, antibody,
tissue, etc.), depends on the natural properties of the molecule
that can be denaturalized under environmental conditions (pH,
temperature or ions)
3. The cells in the biosensor can become intoxicated by other
molecules that are capable of diffusing through the membrane.

2. Recent development topics on biosensors

In biosensor development studies, suitable bioreceptor molecule,
suitable immobilization method and transducer should be selected
firstly. Biology, biochemistry, chemistry, electrochemistry, physics,
kinetics and mass transfer knowledge is reuired for this study. Thus
we can say that developing a biosensor is related with a
interdisciplinary study. Proportional to the technological development
and increase of interdisciplinary studies biosensors are being more
useful and having more usage areas day by day. Recent
development topics which are listed below will be discussed in this
chapter:
 Electrochemical biosensor
Fiber-optic biosensor
Carbon Nanotube
Protein Engineering for biosensors
Wireless Biosensors Networks

2.1 Electrochemical biosensors

Bioelectroanalysis with electrochemical biosensors is a new area in
rapid development within electro analysis. In biosensor development
studies, suitable bioreceptor molecule, suitable immobilization
method and transducer should be selected firstly Bio electroanalytical
sensors permit the analysis of species with great Specificity, very
rapid, sensitive, highly selective and cheap cost in principle. They can
be used in clinical analysis in on-line control processes for industry or
environment, or even in vivo studies [6]. The difference between
biosensor and physical or chemical sensors is that its recognition
element is biological.
The investigated bioelectrochemical reaction would generate a
measurable current (amperometric detection), a measurable
potential or charge accumulation (potentiometric detection) or
measurable conductivity change of a medium (conductometric
detection) between electrodes. When the current is measured at a
constant potential this is referred to as amperometry. If an electrical
current is measured while controlled variations of the potential is
being applied, this is named as voltammetry.
Potentiometric, amperometric and conductometric measurement
techniques forms the kinds of electrochemical biosensors.
Potentiometric sensors have an organic membrane or surface that is
sensitive to an analyte. The reaction between them generates a
potential (emf) proportional to the logarithm of the electrochemically
active material concentration. This potential is compared with the
reference electrode potential.
Enzyme immobilized electrodes reacts with substrate and products
are detected by electrodes. Amperometric sensors measure the
current change resulted by chemical reaction of electroactive
materials while a constant potential is being applied. The change of
the current is related to the concentration of the species in solution.
Generally biological compounds (glucose, urea, cholesterol, etc.) are
not electroactive, so the combination of reactions to produce an
electroactive element is needed. This electroactive element leads a
change of current intensity. This change is proportional to the
concentration of analyte.
Conductometric biosensors can measure the change of the electrical
conductivity of cell solution. Most reactions involve a change in the
composition of solution. Thus, conductometric biosensors can detect
any reactive change occuring in a solution.
Electrochemical biosensors have advantages that they can sense
materials without damaging the system [7]. The use of biosensors for
industrial and environmental analysis [8] is very important. The
control of food manufacturing processes, evaluation of food quality,
control of fermentation processes and for monitoring of organic
pollutants are some of the applications of biosensors. The present
popularity of analytical biosensors is due to their specific detection,
simple use and low cost. For example, an electrochemical biosensor
can be used to detect Salmonella and E. coli O157:H7 in less than
90 min. [7]. Electrochemical biosensor studies are performed with
electrochemical cells.

Electrochemical Cells
An electrochemical cell is used in electrochemical sensor studies.
The electrodes themselves play an important role in the performance
of electrochemical biosensors. The electrode material, its surface
modification or its dimensions effects the detection ability of the
electrochemical biosensor. There are three kinds of electrodes in the
electrochemical cell:
Working electrode
Reference electrode
Auxilary (counter) electrode
Fig. 1. Electrochemical cell.

Auxilary (Counter) Electrode:

In a two-electrode system, when a known current or potential is
applied between the working and auxiliary electrodes, the other
variables may be measured. The auxiliary electrode functions as a
cathode whenever the working electrode is operating as an anode
and vice versa. The auxiliary electrode often has a surface area
much larger than that of the working electrode. The half-reaction
occurring at the auxiliary electrode should occur fast enough not to
limit the process at the working electrode. The potential of the
auxiliary electrode is not measured against the reference electrode
but adjusted to balance the reaction occurring at the working
electrode. This configuration allows the potential of the working
electrode to be measured against a known reference electrode.
Auxiliary electrode is often fabricated from electrochemically inert
materials such as gold, platinum or carbon.

Working Electrode:
It is the electrode on which the reaction occurs in an electrochemical
system [9, 10, 11]. In an electrochemical system with three
electrodes, the working electrode can be referred as either cathodic
or anodic depending on the reaction on the working electrode is a
reduction or an oxidation. There are many kinds of working
electrodes. Glassy carbon electrode, screen printed electrode, Pt
electrode, gold electrode, silver electrode, Indium Tin Oxide coated
glass electrode, carbon paste electrode, carbon nanotube paste
electrode etc.
Screen printed electrodes are prepared with depositing inks on the
electrode substrate (glass, plastic or ceramic) in the form of thin
films. Different inks can be used to get different dimensions and
shapes of biosensors. Screen-printed electrochemical cells are
widely used for developing amperometric biosensors because these
biosensors are cheap and can be produced at large scales. This
could be potentially used as disposable sensor that decreases the
chances of contamination and prevents loss of sensitivity. Figure 3.
exhibits an electrochemical biosensor as screen printed electrode.
Performance factors of an electrochemical biosensor are: Selectivity,
response time, sensitivity range, accuracy, recovery time, solution
conditions and the life time of the sensor.

Fig. 3. Electrochemical biosensor as screen printed electrode.

Electrochemical impedance spectroscopy (EIS) is a technique well

suited for evaluating coating permeability or barrier properties for
corrosion control of steel structures based on the electrical
resistance of the coating. EIS has been widely used in the lab to
determine coating performance and to obtain quantitative kinetic and
mechanistic information on coating deterioration [15].

Immobilization methods
Electrochemical detection techniques use predominant enzymes.
Because enzymes have specific binding capabilities and biocatalytic
activity. Some of the other biorecognition elements are antibodies,
nucleic acids, cells and micro-organisms. Biorecognition elements
should be immobilized on the electrode surface. Adsorption,
microencapsulation, entrapment, covalent attachment and cross
linking methods Are the most well known immobilization methods.
Adsorption methods:
1. Physical adsorption (physisorption) and
2. Chemical adsorption (chemisorption).
Physisorption is weaker than chemisorption. Adsorption is the
simplest way for immobilization of organic material, however the
bonding is weak and life time of electrode is short.
Microencapsulation method is more reliable for adsorption. In this
method, an inert membrane traps the biologic material on the
working electrode. Most used membranes are cellulose acetate,
collagen, gluter aldehyde, chitosan, nafion, polyurethanes, etc..
In entrapment method, generally a solution of polymeric materials
are prepared containing biologic material that will be entrapped onto
the working electrode. The solution is coated on the electrode with
various coating methods. Starch gels, nylon and conductive
polymers such as polyaniline or nafion are used for.
Covalent attachment immobilization is important particularly for the
advantage that the enzyme is not been released from the electrode
surface when it is used. However, covalent bonding should not
decompose or hide the active site of the enzyme. The functional
groups that may take part in this binding are NH 2, CO2H, OH,
C6H4OH and SH groups. [15].
Cross linking is bonding two or more molecules by covalent bonds. In
cross-linking method bifunctional agents such as gluteraldehyde are
used to bind the biological materials. The disadvantage of this
method is high ratio of enzyme activity loss.

2.2 Fiber optic biosensor

The optical fiber is flexible and has small wires generally made out of
glass or plastic in different configuration, shape, and size. It can
transmit light signals for long distances with minimum lost value. The
optical fiber is convenient for harsh and hazardous environments,
because of their remarkably strong, flexible and durable structures. It
is non-electrical; therefore, it can be used in various damaged
electric current applications. Optical fibers are commonly used
because of high quality and its low cost for sensing applications.
Particularly, the main attractive properties of optical fibers can permit
transmission of multiple signals synchronously and by this means it
can obtain multiple capabilities for sensing of analyte [17].
Figure 5 exhibits the optic fibers model that is containing a core and
coating. Their refractive indices are n1 and n2, respectively as shown
in Figure 6. The core and cladding interference act as mirror because
of their different refractive indices (Fig 7a) [17].
Fig. 5. Optic Fiber
The core and cladding parts play a very important role particularly on
the light transmission. Their refractive indices are n 1 and n2,
respectively (Fig 5). In the Fig 6a the core and cladding interference
act as mirror because of their different refractive indices. The series
of internal reflections transmit the light from one end of the fiber to
the other one [17].

Fig. 6. TIR Principle

Fig. 7. The lateral section of Fiber Optic and reflection of signal in

Fiber Optic
 Fiber Optic is used in optical fiber biosensors that
measure some biomolecules such as proteins, nucleic acids etc.)
Because of the attractive properties of fiber optic biosensor such as
low cost, efficiency, accuracy, these take place of literature and they
are preferred in many applications.
The Fiber Optic biosensor provides alternative measurements
method to conventional methods for determination of biological
species.

Fig. 8. The Fiber Optic Biosensor

The Fiber Optic Biosensor have some advantages and

disadvantages which are shown below.
The Advantages of Fiber Optic Biosensor [18, 19]
1. There is no need reference electrode in the system
2. It can be easily moved, because there is no reagent in contact of
any optical fiber
3. There are no electrical safety hazards and electrical interference
4. It is less dependent than temperature compared with electrode
5. It can be found in-vivo measurement applications because of
easy miniaturization
6. Multiple analytes can be determined thanks to guide the light in
different wavelengths at the same time.
7. It can be used for the most of chemical analytes because of its
spectroscopic properties.
The Disadvantages of Fiber Optic Biosensor
1. The life time of the reagents can be short under incident light
2. Because of the diffusion of analytes, it may cause slow response
time
3. Fiber Optic Biosensor only works for spesific reagent.
4. Optimized commercial accessories have limited availability when
using them with optical fibers.

2.3 Carbon nanotube biosensor

The most of the scientists have claimed that a coupling of material
science and biology in the nanosize will have a remarkable effect on
the many fields of science and technology. Particularly in the biology
field, nanosize is very important scale because many important
biomacromolecules structures are in the range of 1-1000nm. [23]
Because of these reasons, the focus is on nanostructured materials.
It helped develop the unique properties of new devices and sensors.
These nanostructured materials have good chemical sensitivity,
biocompatibility, and good electrical sensitivity with changes of
chemical composition. The sensitive materials have played a
significant role for the chemical and biological sensor because of
their sizes which are close to biomolecules.
The performance and improvement of biosensors highly depend on
the materials. Moreover the chosen materials of making transducer
are directly related to their physicochemical characteristics.
The carbon materials such as carbon nanotubes (CNTs) are used in
making biosensor.
CNT’s are well ordered and hexagonal arrangements of Carbon
atoms which have been rolled into tubes. It can be considered as the
cylindrical graphite layer or layers which have nanometer scale of
diameter. Therefore, it can be classified as single wall (SWNT) and
multiwall carbon nanotube (MWNT) as structural. The diameter of
SWNT is approximately 0.4-2 nm and the other one is 2-100 nm.

Fig. 10. Single and Multi- walled Carbon Nanotubes, respectively.

They can also be metallic conducting or semiconducting carbon

nanotubes which change with geometrical structure. The chiral
angle, which determines the twisting value of CNT, play important
role on the conductivity of CNTs. It can be called as zig zag,
armchair and chiral structure (Fig. 12)

Fig. 12. Classification of CNTs, a) Arm-chair, b) Zig-zag, c) Chiral

CNTs

They have some advantages such as small size, high strength

(approximately 100 times higher than the strength of steel), high
electrical (approximately 100 times greater than for cupper wires)
and thermal conductivity (higher than diamond), high specific surface
area, simple preparation, less power, long term stability, good
reproducibility, fast response etc. Therefore, CNT has better
properties than other materials which are used in making biosensor
and the researchers are interested in using CNT for next-generation
of sensors. Because of these properties the researchers consider
that CNT biosensor has the potential of revolutionizing the sensor
area.

The advantages of CNT biosensors help it to perform better in many

of the biomedical sensing applications. Therefore, CNT-based
biosensors are highly suitable as implantable sensors.
In some studies, the dynamic parameters of biosensor such as
response time and sensitivity with either carbon nanotube or without
carbon nanotube were investigated. Decreasing of response time
and increasing of sensitivity because of increasing electron transfer
rate in the presence of the CNTs were reported [44-47]. Moreover,
CNT’s have excellent catalytic activity which decrease their oxidative
potential to avoid fouling problems. The enzymes can be chemically
immobilized to materials in the presence of CNT. For all these
advantages of CNT biosensors are very convenient device to detect
biological molecules.
Future Biosensors Directions:
Miniaturization of developed biosensors will be important in the
future. Because miniaturization is required for small electrodes, for
example measurements in vivo. Another future approach is the
combination of biological materials with a silicon chip because it
seems to be the most comprehensive integration between biology
and electronics [50]. Nanostructures will be important new
components in recently developed electrochemical biosensors:
Nanowires, carbon nanotubes, nanoparticles and nanorods are some
of the familiar objects that are crucial elements of future
bioelectronics devices and biosensors [52].
sensor network. Since the fiber optic cables have multi wires, data is
transmitted very fast. In wired sensor networks require fast
transmission; electromagnetic waves are produced. In those
networks; each member can send data to network because each
member has unique MAC address. Addressed networks can decide
whether the data travelling data is belonging to it or not with the help
of the MAC number. Each member can manage it through the
software on the network [56].