1. M.Prasad Naidu
MSc Medical Biochemistry,
2. Biosensor is an analytical device for the detection of
an analyte that combines a biological component with
a physico chemical detector
3. " A biosensor is a device that detects, records, and transmits
information regarding a physiological change or the presence of
various chemical or biological materials in the environment
More technically, a biosensor is a probe that integrates a
biological component, such as a whole bacterium or a biological
product (e.g., an enzyme or antibody) with an electronic
component to yield a measurable signal.
Biosensors, which come in a large variety of sizes and shapes, are
used to monitor changes in environmental conditions.
They can detect and measure concentrations of specific bacteria
or hazardous chemicals; they can measure acidity levels (pH). In
short, biosensors can use bacteria and detect them, too.
4. the sensitive biological element (biological material
(e.g. tissue, microorganisms, organelles, cell
receptors, enzymes, antibodies, nucleic acids, etc.), a
biologically derived material that interacts (binds or
recognises) the analyte under study. The biologically
sensitive elements can also be created by biological
5. the transducer or the detector element (works in a
optical, piezoelectric, electrochemical, etc.) that
transforms the signal resulting from the interaction of
the analyte with the biological element into another
signal (i.e., transducers) that can be more easily
measured and quantified;
6. Biosensor reader device with the associated electronics
or signal processors is primarily responsible for the
display of results
This sometimes accounts for the most expensive part
of the sensor device.
The readers are usually custom designed and
manufactured to suit the different working principles
7. First generation: the two components (biocatalyst &
tranducer) may be easily separated & both may remain
functional in the absence of the other
Second generation :the two components interact in a
more intimate fashion & removal of one of the two
components affects the usual functioning of the other
Third generation : the biochemistry & where the
electrochemistry occurs at a semiconductor ,the term
biochip may be applied to describe such instruments.
8. First generation instruments
The rate of consumption of the substrate o2 can be
measured by its reduction at a platinum cathode
The rate of production of the product H2O2 can be
measured by its oxidation at a platinum anode
The rate of production of the product gluconic acid
can be measured using a pH electrode
9. YSI MODEL 23
10. Second generation instruments
SECOND GENERATION INSTRUMENTS can be
constructed by designing an electrode surface that is
capable of capturing electrons which are usually
transferred in the oxidation reduction reactions.
13. Third generation instruments
These instruments involve the most intimate
interactions of the biocatalyst and transducer
A glucose biosensor operating on the principle of Exac
Tech meter but in which the enzyme was directly
reduced at the electrode surface (obviating the need
for a mediator) is an example of such an instrument
14. Cell based biosensors
Immobilised whole cells or tissues are used to produce
More recent immobilisation techniques have intended
to use gentler physical methods such that cell viability
The advantage of this is that such cells may be
involved in converting substrate into product via a
complex multi enzyme pathway
Without having to immobilise each of the enzymes &
then provide them with expensive coenzymes
15. Eg.,Nocardia erythropolis immobilised in poly acrylamide
on an oxygen electrode
Cholesterol+ O2 cholest-4-en-3-one+ H2O2
The oxygen electrode measures the rate of oxygen uptake &
this can be related to the cholesterol content of the
No requirement for a
They have longer response
times than do enzyme
Cells contain many
enzymes.Hence, care has
to be taken to ensure
selectivity of response
Time taken for cell based
biosensor to return to base
line potential after use is
17. Enzyme immunosensors
Several kinds of enzyme immunosensors have been
They combine the molecular recognition properties of
antibodies with the high sensitivity of enzyme based
The enzyme is used as a marker as it reacts with its
substrate,giving changes that can be detected by a
There is similarity between such methods & ELISA
18. A similar assay can be carried out for hCG .
Catalase is used to label hCG & oxygen evolution
noted by oxygen electrode
In attempts to construct enzyme
& fluorescence principles are exploited because of
their great sensitivity
A luminiscent immunoassay with catalase has been
used to detect human serumalbumin at only 1 ng/cm3
19. Enzyme based biosensors are used in different
analysers for quantification of glucose (PO2 electrode)
,urea, creatinine etc where the enzyme is immobilized
on the sensor.
Affinity sensors have immobilized molecules with
specific high affinity binding properties like binding
proteins, antibodies, aptamers (DNA SENSORS)
20. Oxygen electrode
The reaction occuring at the anode is 4Ag+4Cl---
The Overall electrochemical process is
22. Schematic diagram showing the
main components of biosensor
23. The biocatalyst (a) converts the substrate to product.
This reaction is determined by the transducer (b)
which converts it to an electrical signal.
The output from the transducer is amplified (c),
processed (d) and displayed (e).
24. Principles of detection
Ion channel switch
Many optical biosensors based on the principle of surface
plasma resonance(SPR) are evanescent wave techniques
This utilises a property of gold & other materials
specifically that a thin layer of gold on a high refractive
index glass surface can absorb light producing electron
waves (surface plasmons) on the glass surface.
This occurs only at a specific angle & wave length of
incident light and is highly dependent on the surface of
gold , such that binding of a target analyte to a receptor on
the gold surface produces a measurable signal
26. Interferometric reflectance imaging
The Interferometric Reflectance Imaging Sensor (IRIS)
was developed by the Unlu research group at Boston
University for the purpose of label-free biosensing.
Using simple lenses and low-powered, coherent
LED’s, the device offers exquisite sensitivity and
reproducibility and is able to image with remarkable
resolution beyond the classical diffraction limit.
This relatively cheap solution also presents minimal
hazards when compared to a laser illumination source.
27. Practical uses of this device include the detection of
bacterial and viral infections in underdeveloped countries.
When pathogen specific growth factors are introduced
into a microarray, only spots with the targeted pathogens
will grow and increase in concentration.
In turn, this dictates a change in the reflected intensity
compared to pre-growth. Thus, by measuring how
reflectance changes over time, unknown pathogens and
their growth rates can be easily characterized and
28. Electro chemical biosensors
Electrochemical biosensors are normally based on catalysis
of reaction that produces or con sumes electrons
(such enzymes are rightly called redox enzymes).
The sensor substrate usually contains three electrodes;
a reference electrode, a working electrode and a counter
The target analyte is involved in the reaction that takes
place on the active electrode surface, and the reaction may
cause either electron transfer across the double layer
(producing a current) or can contribute to the double layer
potential (producing a voltage).
29. We can either measure the current (rate of flow of
electrons is now proportional to the analyte
concentration) at a fixed potential or the potential can
be measured at zero current (this gives a logarithmic
Further, the label-free and direct electrical detection
of small peptides and proteins is possible by their
intrinsic charges using biofunctionalized ion-sensitive
30. All biosensors usually involve minimal sample
preparation as the biological sensing component is
highly selective for the analyte concerned
They enable the detection of analyte at levels
previously only achieved by HPLC & MS & with out
rigorous sample preparation
31. Ion channel switch
The use of ion channels has been shown to offer highly
sensitive detection of target biological molecules. By
imbedding the ion channels in supported or tethered
bilayer membranes (t-BLM) attached to a gold electrode,
an electrical circuit is created
. Capture molecules such as antibodies can be bound to the
ion channel so that the binding of the target molecule
controls the ion flow through the channel. This results in a
measurable change in the electrical conduction which is
proportional to the concentration of the target.
32. An Ion Channel Switch (ICS) biosensor can be created
using gramicidin, a dimeric peptide channel, in a
tethered bilayer membrane.
One peptide of gramicidin, with attached antibody, is
mobile and one is fixed.
The magnitude of the change in electrical signal is
greatly increased by separating the membrane from
the metal surface using a hydrophilic spacer.
33. Ion channel switch
Ion channels open Ion channels close
Piezoelectric sensors utilise crystals which undergo an elastic
deformation when an electrical potential is applied to them.
An alternating potential (A.C.) produces a standing wave in the crystal
at a characteristic frequency.
This frequency is highly dependent on the elastic properties of the
crystal, such that if a crystal is coated with a biological recognition
element the binding of a (large) target analyte to a receptor will
produce a change in the resonance frequency, which gives a binding
In a mode that uses surface acoustic waves (SAW), the sensitivity is
greatly increased. This is a specialised application of the Quartz crystal
microbalance as a biosensor.
35. Surface attachment of biolocal
An important part in a biosensor is to attach the biological elements
(small molecules/protein/cells) to the surface of the sensor (be it
metal, polymer or glass).
The simplest way is to functionalize the surface in order to coat it with
the biological elements. This can be done by polylysine, aminosilane,
epoxysilane or nitrocellulose in the case of silicon chips
Another group of hydrogels, which set under conditions suitable for
cells or protein, are acrylate hydrogel, which polymerize upon radical
initiation. One type of radical initiator is aperoxide radical, typically
generated by combining a persulfate with TEMED (Polyacrylamide
gel are also commonly commonly used for protein electrophoresis)
36. Applications of biosensors
Glucose monitoring in diabetes patients ←historical
market driver related targets
Remote sensing of airborne bacteria e.g. in counter-
37. Invivo biosensors
Invivo miniaturized sensors are being developed for
measurement of saO2,pH etc.
Implantable subcutaneous glucose sensors are also
being used to adjust the dose of insulin
Intravascular sensors that release nitric oxide have
been developed to decrease the possibility of
38. Detection of pathogens
Determining levels of toxic substances before and
Detection and determining of organophosphate
Routine analytical measurement of folic
acid, biotin, vitamin B12 and pantothenic acid as an
alternative to microbiological assay
Determination of drug residues in food, such
as antibiotics and growth promoters, particularly
meat and honey.
Drug discovery and evaluation of biological activity of
Protein engineering in biosensors
Detection of toxic metabolites such as mycotoxins
39. Biosensors in food analysis
There are several applications of biosensors in food analysis. In
food industry optic coated with antibodies are commonly used to
detect pathogens and food toxins. The light signal system in
these biosensors has been fluorescence, since this type of optical
measurement can greatly amplify the pathogens.
A range of immuno- and ligand-binding assays for the detection
and measurement of small molecules such as water-soluble
vitamins and chemical contaminants (drug residues) such
as sulfonamides and Beta-agonists have been developed for use
on SPR based sensor systems, often adapted from
existing ELISA or other immunological assay. These are in
widespread use across the food industry.
40. Detecting Cancer and Health
Tuan Vo-Dinh of Oak
NL) (left) and Bergein
Overholt and Masoud
Panjehpour, both of
Thompson Cancer Survival
Center of Knoxville, have
developed a new laser
technique for nonsurgically
determining whether tumors
in the esophagus are
cancerous or benign.
41. Of these biosensors, the most publicized is the optical
biopsy sensor developed by Tuan Vo-Dinh in collaboration
with medical researchers at Thompson Cancer Survival
Center in Knoxville.
This sensor can tell whether a tumor in the esophagus is
cancerou s or benign.
In the past, determining accurately whether a patient has
cancer of the esophagus has required surgical biopsy.
However, laser-based fluorescence method has eliminated
the need for biopsy, reducing pain and recovery time for
42. Laser light of the appropriate wavelength is directed
to the inner surface of the esophagus by means of a
fiber-optic device that is swallowed by the patient.
The epithelial cells and tissue inside the esophagus
fluoresce when exc ited by the laser light. When the
esophagus interior is illuminated with blue light [410
nanometers (nm)], the normal tissue emits light at
wavelengths different from those emitted by the
43. the spectral properties of the light at wavelengths
ranging from 400 to 700 nm can be analyzed at various
positions in the esophagus by the soft ware developed.
Emissions from normal cells and cancer cells can be
distinguished quite accurately; the difference is
expressed as the differential normalized fluorescence
Tests on more than 200 patients show that, compared
with the results of surgical biopsies, laser fluorescence
diagnosis is accurate in over 98% of the cases.
44. Medical telosensors
telesensor" chip on a
fingertip can measure
and transmit body
45. Medical telesensors
A chip on our fingertip may someday measure and
transmit data on body temperature.
An array of chips attached to our body may provide
additional information on blood pressure, oxygen
level, and pulse rate.
This type of medical telesensor, which is being
developed at ORNL for military troops in combat
zones, will report measurements of vital functions to
46. The goal is to develop an array of chips to
collectively monitor bodily functions. These
chips may be attached at various points on a
soldier using a nonirritating adhesive like that
used in waterproof band-aids
These medical telesensors would send
physiological data by wireless transmission to
an intelligent monitor on another soldier's
The monitor could alert medics if the data
showed that the soldier's condition fit one of
five levels of trauma.
47. The infra red microspectrometer
at ORNL can be used for
blood chemistry analysis,
gasoline octane analysis,
industrial process control,
aircraft corrosion monitoring,
and detection of chemical
48. ORNL has developed a sensitive
detector for monitoring changes in
the body's concentrations of
calcium ions. It may be useful in
diagnosing disease or exposure to
chemical warfare agents. This
biosensor consists of an optical fiber
to which is attached a synthesized
hybrid molecule. One half of the
hybrid molecule binds calcium ions
and the other half fluoresces when
calcium ions are bound to the
49. Blood pressure and pulse rate may be measured by chips
designed to detect pressure changes.
Unlike a glass fiber, a silicone fiber is flexible—it can be
squeezed or stretched, and the amount of compression or
expansion can be measured by changes in light transm
ission through the fiber.
Thus, silicone fibers embedded in roads can be used to
If a silicone fiber on a chip can sense pressure at various
positions in the body, it may be used for monitoring blood
pressure, pulse rate, breathing (chest expansion), knee
bending during physical rehabilitation, and foot pressure
50. Aging, diseases such as diabetes and Alzheimer's, and chemical
warfare agents cause changes in metal ion concentrations in the
If these changes could be detected and measured, the
information could provide clues about changes in disease states a
nd exposure to toxins.
Tuan Vo-Dinh and his coworkers have developed a biosensor
using a glass optical fiber and a hybrid molecule .
One half of the hybrid molecule binds calcium ions and the
other half fluoresces when calcium ions are bo und to the
molecule. By attaching this molecule to the end of a very small
diameter optical fiber, the conc of calcium ions in a solution can
51. ROLE OF
52. Point of care testing is a laboratory testing conducted
close to the site of patient care.
Point of care testing is also commonly described as
ancillary ,bed-side ,near-patient, satellite,remote &
53. Role of biosensors in the
management of diabetes mellitus
Self monitoring of blood glucose(SMBG)
SMBG is the most important day to day metabolic
parameter that must be assessed by the person
affected with diabetes
All insulin treated patients regardless of type 1 or type
2 DM should optimally perform SMBG three or more
The most modern SMBG strips can be used with any
whole blood specimen, whether arterial, venous or
capillary & are suitablefor use in neonates
54. A common example of a commercial biosensor is the blood
glucose biosensor, which uses the enzyme glucose
oxidase to break blood glucose down.
In doing so it first oxidizes glucose and uses two electrons
to reduce the FAD (a component of the enzyme) to FADH2.
This in turn is oxidized by the electrode (accepting two
electrons from the electrode) in a number of steps.
The resulting current is a measure of the concentration of
glucose. In this case, the electrode is the transducer and
the enzyme is the biologically active component.
55. The newest blood glucose detection systems use an
electrochemical biosensor based on a glucose
dehydrogenase reaction that is not affected by oxygen
Glucose dehydrogenase catalyses the formation of
glucono-d-lactone from glucose while NAD+ is
reduced to NADH
The glucose dehydrogenase assays are highly specific
for glucose & show good agreement with hexokinase
Glucose +NAD+ D-glucono-d-lactone+
56. Continuous glucose monitoring system(CGMS)
CGMS involves placing under the skin a small needle
that is attached by a wire to a control module
Typically the needle is inserted under the skin
overlying the abdomen
The needle contains the electrochemical micro
electrode sensor that is thinly coated with glucose
oxidase beneath a biocompatable membrane
The measurement of the interstitial fluid glucose is
changed into an electronic signal & stored in a pager
sized control module,which can be worn on the
The patient does not have access to the CGMS glucose
results while they are wearing the device
57. A glucose reading is taken every 10 sec & the
measurements are averaged and recored (288
measurements per day)
In studies of anesthetized dogs ,there was a 5-12 min
delay between changes in blood glucose& interstitial
fluid glucose levels.
The researchers concluded that ‘differences between
plasma & interstitial fluid glucose will not be a
significant obstacle in advancing the use of interstitial
fluid as an alternative to blood glucose measurements’
58. Biochips: a new generation of biosensors using DNA
probes (DNA Biochip) have been developed
Probe recognition is based on the molecular hybridization
process, which involves the joining of a strand of nucleic
acid with a complementary sequence.
Biologically active DNA probes are directly immobilized
on optical transducers which allow detection of Raman,
SERS, or fluorescent probe labels.
DNA biosensors could have useful applications in areas
where nucleic acid identification is involved.
The DNA probes could be used to diagnose genetic
susceptibility and diseases.
The Biochip using antibody probes has recently been
developed to detect the p53 protein system.
59. Using methods to form short sequences from the
DNA of interest, DNA fragments are produced that
have one of the letters at their end and that differ
acco rding to their sizes.
As a result, DNAs containing 400 to 600 letters can
be sequenced accurately.
However, many hours are required to prepare the
fragments and separate them by size (using gel
Using this method, the sequences of millions of
DNA letters have been determined, enabling the
identification of the site of a genetic mutation that
causes such diseases as sickle cell anemia,
Huntington's disease, fragile X syndrome (a serious
type of mental retardation), and several hundr ed
other inherited diseases or traits.
60. Serg probes
ORNL's surface-enhanced Raman gene (SERG) probes
can locate free DNA molecules that have hybridized to
other DNAs fixed on a surface.
The technique has use in medi
cine, forensics, agriculture, and environmental
61. Serg probes
62. Another hybridization method being developed at Oak
Ridge uses a process called surface-enhanced Raman
Hybridized DNA is transferred from the nylon membrane
to a glass strip coated with tiny silver spheres.
The dye labels attached to the DNA bases have unique
Raman infrared spectra, but the normally weak Raman
lines are greatly enhanced by the presence of the silver
This enhancement allows DNA bases to be detected at
sufficient sensitivity to be useful for sequenci ng studies.
63. DNA processing & analysis chip
Conceptional design of
DNA processing and
analysis chip that Mike
Ramsey and Bob Foote
64. To determine which DNA fragments make up a
fingerprint, they must be separated and identified.
The "lab on a chip" developed by Mike Ramsey and
colleagues has been adapted to perform such
separations within a few minutes—much faster than
standard gel procedures.
Like microcircuits and computers operating in
parallel, these chemical separations chips can be used
in parallel for DNA analysis.
65. In one application, liquids containing DNA and a
restriction enzyme are injected into different
chambers etched into the chip.
Electric fields pump the liquids through a microscopic
channel into a reaction chamber, where the enzyme
cuts the DNA into pieces of different lengths.
The DNA snippets are then electrically pumped to the
separation channel, wher e they are tagged with
fluorescent dyes for detection.
66. The DNA fragments of various sizes are sorted in a liquid
containing fibrous strands of a polymer. The DNA through
fragments get tangled with the polymer strands, which
slow them down as they pass.
Small chunks of DNA find their way through the tangled
web faster than the larger ones, so separation results.
As the fragments ar e separated, they are illuminated with a
laser light, causing them to fluoresce.
The detected light intensities are fed to a computer, which
sorts through signals from separated fragments to provide a
NANOSENSORS: the development of nano-biosensors and in situ
intracellular measurements of single cells using antibody-based
nanoprobes has recently been reported.
The nano-scale size of this new class of sensors also allows for
measurements in the smallest of environments. One such environment
that has evoked a great deal of interest is that of individual cells.
Using these nanosensors, it is possible to probe individual chemical
species and molecular signalling processes in specific locations within
it has been shown that insertion of a nano-biosensor into a
mammalian somatic cell not only appears to have no effect on the cell
membrane, but also does not effect the cell's normal function.
68. Bruce Jacobson (left) and Carl
Gehrs, manager of ORNL's
Biotechnology, examine a
newly fabricated temperature
control block for a
genosensor chip designed by
Mitch Doktycz. The
genosensor chip can detect
specific DNA sequ ences.
69. Atomic force microscopy
This atomic force
microscopy image shows
two knots, or "protein
bumps," formed on the
DNA thread. Each knot
results when a mutant
enzyme binds to, rather
than cleaves, the DNA
70. The magic scissors enzyme, or restriction
endonuclease, cuts a DNA sequence each time it
occurs in the strand.
But the mutant form of this enzyme simply binds
to, rather than cleaves, the DNA strand. The AFM
can image the resulting knot, or protein bum p,
formed on the DNA thread.
ORNL researchers can identify where the enzyme
binds to the DNA within 100 base pairs, which is a
very high resolution.
They've shown that the distance from one bump to
the next, as imaged by the AFM, was exactly as
predicted from enzyme cutting experiments
analyzed by gel electropho resis.
71. beams and mirrors can
determine the shapes of
human body parts. Its accuracy
could facilitate the creation of
clothes that fit.
72. Perhaps the most unusual of ORNL's biosensors is a new technique to
measure human body surfaces. Such measurements, called
anthropometry, are used by tailors, artists, and scientists.
One of the finest minds in science to take a strong interest in anth
ropometry was Leonardo da Vinci, who drew the famous Proportions
of the Human Figure some 500 years ago. An ORNL scientist who has
moved the field forward is Judson Jones of the Computer Science and
Mathematic s Division.
He has developed a technique using laser beams and mirrors to
determine the shape of human body parts. He measures the topology
of a solid surface, using amplitude-modulated laser radar, which
measures arc lengths along complex and oft en inaccessible body
Because laser radar measures the phase and amplitude of a reflected,
modulated laser beam, only one optical path is required between the
sensor and the subject. Multiple images are combined by integrating
information from different virtual viewpoints, any number of which
can be created with strategically positioned mirrors.
Arc lengths along arbitrary contours, surface areas, and volume
estimates all become possiusing ble. The accuracy of the
measurements is within 1 mm. Be cause creating "clothes that fit" may
be accomplished a single camera with several mirrors, a blue jeans
manufacturer has shown interest in this methodology.
73. Light emissions from
microspheres and bacteria are
seen through a fluorescence
microscope. Shown are red-
fluorescing S. aureus bacteria
bound to 6.5-µm spheres and
one yellow orange-
fluorescing E. coli bound to
the larger 10-µm sphere.
74. 100 different types of bacteria can be identified
simultaneous ly because the stained bacteria all would
fluoresce at one wavelength and of light the diameters of
the spheres could be illuminated at another wavelength.
Although all the spheres fluoresce when excited by one
wavelength, the morphological resonances, which look like
saw teeth superimposed on a fluorescence emission
spectrum, can distinguish among diameters of many
This approach satisfies one of today's challenges in
biotechnology: multiplex biosensors to obtain more infor
mation from one sample analysis.
75. In another approach to the use of immunosensors,
microspheres of different sizes are labeled with antibodies
that bind to different bacteria; thus, microspheres of one
size have one particular antibody and microspheres of
another s ize have a different antibody.
The sizes of the microspheres are identified by their
"morphological resonances" (shape-based light emissions
when excited by a laser), and the bacteria that become
bound are detected by the color of fluorescent dye with
which they are stained.
Genetically engineered bacteria can also be useful because of
their ability to "tattle" on the environment.
Such commonly used bacteria have been designed to give off a
detectable signal, such as light, in the presence of a specific
pollutant they like to eat.
They may glow in the presence of toluene, a hazardous
compound found in gasoline and other petroleum products.
They can indicate whether an underground fuel tank is leaking
or whether the site of an oil spill has b een cleaned up effectively.
These informer bacteria are called bioreporters.
77. The key to protecting a military unit or community
from dangerous bacteria is to detect them before they
reach their intended victims. People can then be
warned to leave an area or at least wear protective
gear. Bacteria can be detected using "biosensors.
78. Critters on a chip
Mike Simpson shows the
newly developed "critters
on a chip" in which
signal the presence of
79. "critters on a chip" technology in which light sensor s pick
up and transmit information from chip bacteria that glow
in the presence of trace levels of poisons, explosives, or
These types of biosensors are useful for monitoring efforts
to clean up industrial spills because these light-emitt ing
bacteria can "report" continuously the progress of
Such bioreporters have proven successful using
trichloroethylene, toluene, and various petroleum products
in laboratory tests. They are now being tested on a much
larger scale usi ng a lysimeter.