SEMINAR REPORT
ON
BIOSENSOR
Submitted to
Department of Biotechnology
Deenbandhu Chhotu Ram University of Science &
Technology, Murthal
In partial fulfillment of the requirements for the degree of
BACHELOR OF TECHNOLOGY
IN
BIOTECHNOLOGY
SUBMITTED BY:
POONAM YADAV
(10001008040)
CONTENT
INTRODUCTION
HISTORY
RECENT DEVELOPMENT
FEATURES OF BIOSENSORS
TYPES OF BIOSENSORS
CHARACTERISTICS OF BIOSENSORS
APPLICATION OF BIOSENSORS
DNA BIOSENSOR
ADVANTAGE OF BIOSENSOR
DISADVANTAGE OF BIOSENSOR
INTRODUCTION
A biosensor is an analytical device which converts a biological response into an electrical
signal . The term 'biosensor' is often used to cover sensor devices used in order to
determine the concentration of substances and other parameters of biological interest
even where they do not utilise a biological system directly. This very broad definition is
used by some scientific journals (e.g. Biosensors, Elsevier Applied Science) but will not be
applied to the coverage here. The emphasis of this Chapter concerns enzymes as the
biologically responsive material, but it should be recognised that other biological systems
may be utilised by biosensors, for example, whole cell metabolism, ligand binding and the
antibody-antigen reaction. Biosensors represent a rapidly expanding field, at the present
time, with an estimated 60% annual growth rate; the major impetus coming from the
health-care industry (e.g. 6% of the western world are diabetic and would benefit from the
availability of a rapid, accurate and simple biosensor for glucose) but with some pressure
from other areas, such as food quality appraisal and environmental monitoring. The
estimated world analytical market is about 12,000,000,000 year
-1
of which 30% is in the
health care area. There is clearly a vast market expansion potential as less than 0.1% of
this market is currently using biosensors. Research and development in this field is wide
and multidisciplinary, spanning biochemistry, bioreactor science, physical chemistry,
electrochemistry, electronics and software engineering. Most of this current endeavour
concerns potentiometric and amperometric biosensors and colorimetric paper enzyme
strips. However, all the main transducer types are likely to be thoroughly examined, for
use in biosensors, over the next few years. A successful biosensor must possess at least
some of the following beneficial features:The biocatalyst must be highly specific for the
purpose of the analyses, be stable under normal storage conditions and, except in the case
of colorimetric enzyme strips and dipsticks (see later), show good stability over a large
number of assays (i.e. much greater than 100).The reaction should be as independent of
such physical parameters as stirring, pH and temperature as is manageable. This would
allow the analysis of samples with minimal pre-treatment. If the reaction involves
cofactors or coenzymes these should, preferably, also be co-immobilised with the
enzyme.The response should be accurate, precise, reproducible and linear over the useful
analytical range, without dilution or concentration. It should also be free from electrical
noise.If the biosensor is to be used for invasive monitoring in clinical situations, the probe
must be tiny and biocompatible, having no toxic or antigenic effects. If it is to be used in
fermenters it should be sterilisable. This is preferably performed by autoclaving but no
biosensor enzymes can presently withstand such drastic wet-heat treatment. In either
case, the biosensor should not be prone to fouling or proteolysis.The complete biosensor
should be cheap, small, portable and capable of being used by semi-skilled
operators.There should be a market for the biosensor. There is clearly little purpose
developing a biosensor if other factors (e.g. government subsidies, the continued
employment of skilled analysts, or poor customer perception) encourage the use of
traditional methods and discourage the decentralisation of laboratory testing.
There are three so-called 'generations' of biosensors; First generation biosensors where
the normal product of the reaction diffuses to the transducer and causes the electrical
response, second generation biosensors which involve specific 'mediators' between the
reaction and the transducer in order to generate improved response, and third generation
biosensors where the reaction itself causes the response and no product or mediator
diffusion is directly involved.
The electrical signal from the transducer is often low and superimposed upon a relatively
high and noisy (i.e. containing a high frequency signal component of an apparently
random nature, due to electrical interference or generated within the electronic
components of the transducer) baseline. The signal processing normally involves
subtracting a 'reference' baseline signal, derived from a similar transducer without any
biocatalytic membrane, from the sample signal, amplifying the resultant signal difference
and electronically filtering (smoothing) out the unwanted signal noise. The relatively slow
nature of the biosensor response considerably eases the problem of electrical noise
filtration. The analogue signal produced at this stage may be output directly but is usually
converted to a digital signal and passed to a microprocessor stage where the data is
processed, converted to concentration units and output to a display device or data store.
The key part of a biosensor is the transducer (shown as the 'black box' in Figure 6.)
HISTORY
The first biosensor was invented by Professor Leland C Clark Jnr. and he is known as the
father of the biosensor concept. On 15 April 1956, at a meeting of the American Society for
Artificial Organs during the annual meetings of the Federated Societies for Experimental
Biology, the biosensor invented was named after him as Clark electrode. In 1956, Clark
published his definitive paper on the oxygen electrode. The concept was illustrated by an
experiment in which glucose oxidase was entrapped at a Clark oxygen electrode using
dialysis membrane (Turner, 1996). This biosensor was made from a thin layer of glucose
oxidase (GOx) on an oxygen electrode.
The amount of glucose was estimated by the reduction in the dissolved
oxygenconcentration. Clarks ideas became commercial reality in 1975 with the successful
re-launch (first launch 1973) of the Yellow Springs Instrument Company (Ohio) glucose
analyser based on the amperometric detection of hydrogen peroxide (Turner 1996). In
1963 Garry A. Rechnitz together with S. Katz introduced one of the first papers in the field
of biosensors with the direct potentiometric determination of urea after urease
hydrolysis. At that time the term biosensor had not yet been coined. Thus, these types
of devices were called enzyme electrodes or biocatalytic membrane electrodes. For the
first time, in 1964 enzymes were used as fuel cell catalysts by Yahiro et al. in a glucose/O
2biofuel cell. In 1969 George Guilbault introduced the potentiometric urea electrode.
In 1973 Ph. Racinee and W. Mindt developed a lactate electrode. In 1976 came the first
microbe-based biosensor and finally in 1977 Karl Cammmann introduced the term
biosensor. Lubbers and Opitz coined the term optode in 1975 to describe a fibre-optic
sensor with immobilised indicator to measurecarbon dioxide or oxygen (Turner, 1996).
They extended the concept to make an optical biosensor for C2H5OH by immobilising
C2H5OH oxidase on the end of a fibre-optic oxygen sensor. The idea of building
direct immunosensors by fixing antibodies to a piezoelectric or potentiometric transducer
had been explored since the early 70s, but it was a paper by Liedberg et al. (1980) that
was to pave the way for commercial success. They described the use of surface
plasmon resonance to monitor affinity reactions in real time.
The BI Acore (Pharmacia, Sweden) launched in 1990 is based on this technology (Turner,
1996). In 1979 pioneering work by J. Kulys using artificial redox mediators and in 1984
Cass et al. introduced first ferrocene-mediated amperometic glucose biosensor which
was commercialised by MediSense Inc. in 1987 with a pen-sized meter for home blood-
glucose monitoring.. In 1997 IUPAC introduced for the first time definition for biosensors
in analogy to the definition of chemosensors. An enzymatic glucose/O 2 fuel cell which
wasimplanted in a living plant was presented by Heller and coworkers in 2003. The first H
2 /O 2 biofuel cell based on the oxidation of low levels of H 2 in air was introduced by
Armstrong and coworkers (2006). In 2007 an implanted glucose biosensor (freestyle
Navigator system) operated for five days (Borgmann et al.,2011).
Recently nano-biosensors, implanted biosensors and integrated biosensors are in current
research and development. In the past 40 years various biosensors have been researched
and developed encompassing a wide range of applications but the number of
commercially available biosensors are limited. Hence Biosensor technology is an
upcoming field and with continued progress, we can expect that biosensors can become
simpler and more widely available commercially.
RECENT DEVELOPMENT
Stanford researchers have developed a new biosensor microchip that could significantly
speed up the process of drug development. The microchips, packed with highly sensitive
"nanosensors," analyze how proteins bind to one another, a critical step for evaluating the
effectiveness and possible side effects of a potential medication. A single centimeter-sized
array of the nanosensors can simultaneously and continuously monitor thousands of
times more protein-binding events than any existing sensor. The new sensor is also able to
detect interactions with greater sensitivity and deliver the results significantly faster than
the present "gold standard" method. "You can fit thousands, even tens of thousands, of
different proteins of interest on the same chip and run the protein-binding experiments in
one shot," said Shan Wang, a professor of materials science and engineering, and of
electrical engineering, who led the research effort."In theory, in one test, you could look at
a drug's affinity for every protein in the human body," said Richard Gaster, MD/PhD
candidate in bioengineering and medicine, who is the first author of a paper describing the
research that is in the current issue of Nature Nanotechnology, available online now.
The power of the nanosensor array lies in two advances. First, the use of magnetic
nanotags attached to the protein being studied such as a medication greatly increases
the sensitivity of the monitoring. Second, an analytical model the researchers developed
enables them to accurately predict the final outcome of an interaction based on only a few
minutes of monitoring data. Current techniques typically monitor no more than four
simultaneous interactions and the process can take hours."I think their technology has the
potential to revolutionize how we do bioassays," said P.J. Utz, associate professor of
medicine (immunology and rheumatology) at Stanford University Medical Center, who
was not involved in the research.Members of Wang's research group developed the
magnetic nanosensor technology several years ago and demonstrated its sensitivity in
experiments in which they showed that it could detect a cancer-associated protein
biomarker in mouse blood at a thousandth of the concentration that commercially
available techniques could detect. That research was described in a 2009 paper in Nature
Medicine. The researchers tailor the nanotags to attach to the particular protein being
studied. When a nanotag-equipped protein binds with another protein that is attached to
a nanosensor, the magnetic nanotag alters the ambient magnetic field around the
nanosensor in a small but distinct way that is sensed by the detector.
"Let's say we are looking at a breast cancer drug," Gaster said. "The goal of the drug is to
bind to the target protein on the breast cancer cells as strongly as possible. But we also
want to know: How strongly does that drug aberrantly bind to other proteins in the
body?"To determine that, the researchers would put breast cancer proteins on the
nanosensor array, along with proteins from the liver, lungs, kidneys and any other kind of
tissue that they are concerned about. Then they would add the medication with its
magnetic nanotags attached and see which proteins the drug binds with and how
strongly."We can see how strongly the drug binds to breast cancer cells and then also how
strongly it binds to any other cells in the human body such as your liver, kidneys and
brain," Gaster said. "So we can start to predict the adverse affects to this drug without ever
putting it in a human patient."It is the increased sensitivity to detection that comes with
the magnetic nanotags that enables Gaster and Wang to determine not only when a bond
forms, but also its strength. "The rate at which a protein binds and releases, tells how
strong the bond is," Gaster said. That can be an important factor with numerous
medications."I am surprised at the sensitivity they achieved," Utz said. "They are
detecting on the order of between 10 and 1,000 molecules and that to me is quite
surprising."The nanosensor is based on the same type of sensor used in computer hard
drives, Wang said."Because our chip is completely based on existing microelectronics
technology and procedures, the number of sensors per area is highly scalable with very
little cost," he said. Although the chips used in the work described in the Nature
Nanotechnology paper had a little more than 1,000 sensors per square centimeter, Wang
said it should be no problem to put tens of thousands of sensors on the same footprint.
"It can be scaled to over 100,000 sensors per centimeter, without even pushing the
technology limits in microelectronics industry," he said.Wang said he sees a bright future
for increasingly powerful nanosensor arrays, as the technology infrastructure for making
such nanosensor arrays is in place today. "The next step is to marry this technology to a
specific drug that is under development," Wang said. "That will be the really killer
application of this technology."Other Stanford researchers who participated in the
research and are coauthors of the Nature Nanotechnology paper are Liang Xu and Shu-Jen
Han, both of whom were graduate students in materials science and engineering at the
time the research was done; Robert Wilson, senior scientist in materials science and
engineering; and Drew Hall, graduate student in electrical engineering. Other coauthors
are Drs. Sebastian Osterfeld and Heng Yu from MagArray Inc. in Sunnyvale. Osterfeld and
Yu are former alumni of the Wang Group.
FEATURES OF BIOSENSORS
A biosensor has two distinct types of components:
(a) Biological, e.g., enzyme, antibodies and
(b) Physical, e.g., transducer, amplifier, etc.
The biological component of biosensor performs two important functions.
(a) It specifically recognises the analyte and
(b) It interacts with it in such a manner which produces some physical change detectable
by the transducer.
These properties of the biological component impart on the biosensor its specifically,
sensitivity and the ability to detect and measure the analyte. The biological component is
suitably immobilized on to the transducer. Generally, the correct immobilization of
enzymes enhances their stability. As a result, many enzyme-immobilized systems can be
used more than 10,000 times over a period of several months.
The biological component interacts specifically to the analyte which produces a physical
change close to the transducer surface. This physical change may be:
1. Heat released or absorbed by the reaction (calorimetric biosensors)
2. Production of an electrical potential due to changed distribution of electrons
(potentiometric biosensors).
3. Movement of electrons due to redox reaction (amperometric biosensors).
4. Light produced or absorbed during the reaction (optical biosensors).
5. Change in mass of the biological component as a result of the reaction (acoustic wave
biosensors).
The transducer detects and measures this change and converts it into an electrical signal.
This signal being very small is amplified by an amplifier before it is fed into the
microprocessor. The signal is then processed and interpreted, and is displayed in suitable
units.
Thus, biosensors convert a chemical information flow into an electrical information flow,
which involves the following steps:
(a) The analyte diffuses from the solution to the surface of the biosensor.
(b) The analyte reacts specifically and efficiently with the biological component of the
biosensor.
(c) This reaction changes the physico-chemical properties of the transducer surface.
(d) This leads to a change in the optical or electronic properties of the transducer surface.
(e) The change in optical/electronic properties is measured, converted into electrical
signal which is amplified, processed and displayed.
TYPES OF BIOSENSORS
1. Calorimetric Biosensors: Calorimetric biosensors measure the temperature change of
the solution containing the analyte following enzyme action and interpret it in terms of
the analyte concentration in the solution. The analyte solution is passed through a small
packed bed column containing immobilized enzyme; the temperature of the solution is
determined just before entry of the solution into the column and just as it is leaving the
column using separate thermistors.
2. Potentiometric Biosensors: These biosensors use ion-selective electrodes to convert
the biological reaction into electronic signal. The electrodes employed are most commonly
pH meter glass electrodes (for cations), glass pH electrodes coated with a gas selective
membrane (for CO2, NH, or H2S) or solid state electrodes. Many reactions generate or use
H+ which is detected and measured by the biosensor; in such cases very weak buffered
solutions are used. Gas sensing electrodes detect and measure the amount of gas
produced.
3. Acoustic Wave Biosensors:These are also called piezoelectric devices. Their surface is
usually coated with antibodies which bind to the complementary antigen present in the
sample solution. This leads to increased mass which reduces their vibrational frequency;
this change is used to determine the amount of antigen present in the sample solution.
4. Amperometric Biosensors:These electrodes function by the production of a current
when potential is applied between two electrodes, the magnitude of current being
proportional to the substrate concentration. The simplest amperometric biosensors use
the Clark oxygen electrode which determines the reduction of O2 present in the sample
(analyte) solution. These are the first generation biosensors. These biosensors are used to
measure redox reactions, a typical example being the determination of glucose using
glucose oxidase.
5. Optical Biosensors:These biosensors measure both catalytic and affinity reactions.
They measure a change in fluorescence or in absorbance caused by the products
generated by catalytic reactions. Alternatively, they measure the changes induced in the
intrinsic optical properties of the biosensor surface due to loading on it of dielectric
molecules like protein (in case of affinity reactions). A most promising biosensor involving
luminescence uses firefly enzyme luciferase for detection of bacteria in food or clinical
samples. The bacteria are specifically lysed to release ATP, which is used by luciferase in
the presence of 02 to produce light which is measured by the biosensor.
CHARACTERISTICS OF BIOSENSORS
1. SELECTIVITY: Selectivity is probably the most important feature of a biosensor.
Selectivity means that sensor detects a certain analyte and doesnt react to
admixtures and contaminants. Antigen-antibody interaction has the highest
selectivity, it is analyte-specific.
2. SENSITIVITY: Sensitivity(detection limit) shows the minimal amount (or
concentration) of analyte that can be detected.
3. LINEARITY: linear range is another important characteristic. Linear range is the
range of analyte concentrations in which the sensor response changes linearly with
the concentration.
4. RESPONSE TIME: Response time is time required to analyze the assay.
APPLICATIONS OF BIOSENSORS
Food analysis
Study of biomolecules and their interaction
Drug development
Crime detection
Medical diagnostic (both clinical and laboratory use)
Environmental field monitoring
Quality control
Industrial process control
Detection system for biological warfare agents
Manufacturing of pharmaceuticals and replacement organs
DNA BIOSENSOR
In the future, DNA will find use as a versatile material from which scientists can craft
biosensors. DNA biosensors can theoretically be used for medical diagnostics, forensic
science, agriculture, or even environmental clean-up efforts. No external monitoring is
needed for DNA-based sensing devises. This is a significant advantage. DNA biosensors are
complicated mini-machinesconsisting of sensing elements, micro lasers, and a signal
generator. At the heart of DNA biosensor function is the fact that two strands of DNA stick
to each other by virtue of chemical attractive forces. On such a sensor, only an exact fit
that is, two strands that match up at every nucleotide positiongives rise to a fluorescent
signal (a glow) that is then transmitted to a signal generator.
ADVANTAGES OF BIOSENSORS
biosensors are highly selective
no sample preparation
low detection limits
many instruments are inexpensive and portable
wide range of applications; can be used for medical purposes, food quality control,
environmental monitoring
DISADVANTAGES OF BIOSENSORS
biosensor materials can denature in harsh conditions
auxiliary electrodes very costly and not included in commercially available sensors
affinity biosensors can be expensive and hard to obtain
