BIOSENSOR POWERPOINT

1. Presented by :
Dr. MANJU JHA
II-year Resident,
M.D. Biochemistry
09-10-2012
J.L.N.Medical College,Ajmer.

2. HISTORY19621969197019751975197519761980198219831984198719901992199619982000-
first description of a biosensor of : an amperometric enzyme electrode (Glucose
sensor) by Clark.
first potentiometric biosensor : urease immobilized on an ammonia electrode to
detect urea by Guilbault & Montalva.
ion-selective Field Effect Transistor (ISFET) by Bergveld.
fibre-optic sensor with immobilized indicator to measure carbon-di-oxide or
oxygen by Lubbers & Optiz.
first commercial biosensor (Yellow Spring Instrumental Biosensor) .
first microbe based biosensor (first Immunosensor)..
first bedside artificial pancrease.
first fibre-optic pH sensor for in-vivo blood gases by Peterson.
first fibre-optic based biosensor for glucose.
first surface plasmon resonance (SPR) immunosensor.
first mediated Amperometric biosensor.
Blood Glucose biosensor launched by Medi-sense Exac Tech.
SPR based biosensor by Pharmacia BIA Core.
hand-held blood biosensor by i-STAT..
launching of Gluco-card.
blood glucose biosensor launched by Life-scan Fast Take.
nanotechnology biosensor, chip,quantum dots etc..

3. What is a BIOSENSOR ?
A Biosensor may be defined as :
a compact analytical device having a
biological or biologically-derived sensing
element either integrated within or
intimately associated with a physicochemical transducer which is then detected
by an electronic component and translated
into a measurable electronic signal.

4. IUPAC Definition -
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
electrochemical transducer element.

5. WORKING PRINCIPLE OF A BIOSENSOR
The interaction of the analyte with the bioreceptor is designed to
produce an effect measured by the transducer, which converts the
information into a measurable effect, such as an electrical /
electronic signal.

6. (e)
(f)
(d)
(a)
(b)
(c)
The interaction of the analyte (a) with the bioreceptor, which identifies
the stimulus (b) is designed to produce an effect measured by the
transducer (c), which converts it to an electrical signal. The output from
the transducer is amplified (d), processed (e) and displayed (f).

7. Main components-
A. Biological recognition element-
It is the sensitive biological element or
biological material (tissue ,microorganisms
, organelles , cell
receptors , enzymes,
antibodies, nucleic acids, etc.) or
biomimetic component
that interacts
(binds or recognises) the analyte under
study.
The biologically sensitive elements can also
be created by biological engineering.

8. B. Transducer-
The transducer or the detector element
transforms the signal resulting from the
interaction of the analyte with the biological
recognition element into another signal that
can be more easily measured and quantified.
C. Amplifier, Microprocessor and Display-
Biosensor reader device with the associated
electronics or signal processors that are
primarily responsible for the display of the
results.

9. Biological elements and Transducers commonly used in BIOSENSORSTransducer elements
Biological elements
1. Enzymes
2. Antibodies
3. Hormone receptors
4. Cells
5. Cell organelle
6. Tissues
7. Membranes
8. Micro organisms
9. Nucleic acids
10. Biomimetic materials
1. Electrochemical
Amperometric
Potentiometric
2. Electrical
Conductometric
Ion-sensitive
3. Photochemical
Calorimetric
Fluorescence
Reflectance
4. Optical
Fiber optic
SPR
Luminometric
Fluorometric
5. Piezoelectric
Acoustic
MCL
Ultrasonic
QCM

10. Biological recognition element— (Classification acc to Biological Signalling method)
A. Catalytic biosensor:
kinetic devices that measure steady state
concentration of a ‘tranducer - detectable
species’ formed / lost due to a biocatalytic
reaction.
Monitored quantities arei. rate of product / acid ( ↑ or ↓pH) formation,
ii disappearance / consumption of reactant,
iii. inhibition of reaction ,etc.

11. Biocatalysts used are----
1.EnzymesEnzyme-based biosensors use their catalytic
activity and binding capabilities for specific
detection .
The catalytic activity of the enzymes
provides these types of biosensors with
the ability to detect much lower limits
than with normal binding techniques.
This catalytic activity is related to the
integrity of the native protein structure.

12. Most common – Glucose oxidase, Urease, Alcohol oxidase etc.
Commercial example is GLUCOSE SENSOR using oxidase (GOD) .
Clarks (1962)
Glucose + O2 -- glusoce oxidase  Gluconic acid + H2O2 (1)
H2O2  O2 + 2H+ + 2e-
O2 + 4H+ + 4 e-  2H2O
-and current flows.
(3) at anode
(2) at cathode
There are three measurement routes-pH change (acid production)
- O2 consumption (fluorophore monitor)
- H2O2 production (electrochemical)

13. 2. Micro-organisms –
Microorganisms such as bacteria and fungi
can be used as biosensors to detect specific
molecules or the overall state of the
surrounding environment.
Cell behaviour such as cell metabolism,
cell viability, cell respiration, and bioluminescence can be used as indicators
for the detection.
Furthermore, proteins that are present in
cells can also be used as bio-receptors for the
detection of specific analytes.
Rhodococcus erythropolis (in collagen) used
to measure BOD.

14. 3. Cell / Tissue samples.
Use of cell as a biosensor occurred in 1977 by
Rechnitz .
Rechnitz coupled Streptococcus faecium on the
surface of an ammonia gas sensing membrane
electrode .
This Rechnitz electrode was capable of detecting
the amino acid ARGININE.
4. Organelles – Mitochondra , Cell walls etc.
5. Membranes .

15. 6. Bio-mimetic materials A bio mimetic biosensor is an artificial or
synthetic sensor that mimics the function of a
natural biosensor .
These can include aptasensors , where apta
-sensors use aptamers as the biocomponent.
Aptamers are synthetic single
stranded
nucleic acid that can be designed to
identify
or
recognize
amino-acids,
oligosaccharides , peptides , and proteins .
Aptamers have high affinity , high selectivity,
cheaper & easy to synthesize .

16. B. Affinity biosensors:
device in which receptor molecules bind analyte molecules
causing physicochemical change that is detected by a transducer.
Receptors used are-
1. Antibody / Antigen (Immunosensor) The high specificity between an antibody and
an antigen can be utilized in this type of sensor
technology. Biosensors utilizing this specificity
must ensure that binding occurs under conditions
where nonspecific interactions are minimized .
Binding can be detected either through
fluorescent labelling or by observing a
refractive index or reflectivity changes.
2. Hormone receptor/Antagonist-

17. 3. Nucleic acid -The complementary relationships between nucleic acid
‘bases’ in the DNA form the basis of specificity in
nucleic acid based biosensors.
These sensors are capable of detecting trace amounts of
microorganism DNA by comparing it to a complementary
strand of known DNA.
By unwinding the target DNA strand, adding the DNA
probe, and annealing the two strands , the probe
will hydrolyze to the complementary sequence on the
adjacent strand . If the probe is tagged with a
fluorescent compound , then this annealing can be
visualized under a microscope.
eg. DNA Chip.

18. (Target probe )
(Capture probe)
General DNA biosensor scheme--Target DNA is captured at the recognition layer (A),
and the resulting hybridization is transduced into a measurable electronic signal (B).
It is used for genome mutation detection & clinical diagnosis.

19. Biosensor construction-I.
Recognition element immobilization (Immobilization of biological receptor)-
Biological receptors, i.e. enzymes, antibodies, cells or tissues with
high biological activity, can be immobilized in a thin layer at the
transducer surface by using different procedures.
(a) Entrapment behind a membrane - viscous aqueous solution
trapped by membrane permeable to analyte.
(b) Entrapment of biological receptors within a polymeric matrixMembranes—
Cellophane, Cellulose acetate, Polyurethane membranes .
Gel entrapment–
Agarose, Gelatin, Agar gel , Polyacrylamide gel.
MicroencapsulationEncapsulation inside Liposomes, or absorbed in fine carbon
.
particles that are incorporated in a gel or membrane

20. (c) Entrapment of biological receptors within
self-assembled monolayers (SAMs)
or bilayer lipid membranes (BLMs).
(d) Adsorption:
direct adsorption onto membrane or transducer;
can also be adsorbed onto pre -adsorbed proteins .
(e) Covalent binding (via –COOH, -NH2, -OH ) , or cross
linking (via glutaraldehyde ) to transducer or
membrane surface.
Receptors are immobilized either alone or are mixed with other
proteins, such as bovine serum albumin (BSA), either directly on
the transducer surface, or on a polymer membrane covering it .

21. II. Inner & Outer membranes-These serve three important functions -
(a) Protective barrier --
Membrane prevents large molecules, such as
proteins or cells of biological samples, from
entering and interfering with the reaction layer.
It also reduces leakage of the reacting layer
components into the sample solution.
This function of the outer membrane is
important, for example, for implanted glucose
sensors, since its glucose oxidase is of nonhuman origin and may cause immunological
reactions.

22. (b) Diffusional outer barrier for the substrate –
The thinner the membrane, the shorter the
biosensor response time so such a diffusional
barrier
also
makes the sensor response
independent of the amount of active enzyme
present and improves the sensor response stability.
(c) Biocompatible and biostable surfaces –
Biosensors are subject to modifications when they
are in direct contact with biological tissues or fluid ,
i.e. implanted in vivo or, more generally, in
biologically active matrices, such as cell cultures.

23. BIOCOMPATIBILITY-
If the implantation of the biosensor does
not materially affect
the normal
functioning of the host medium and if
the medium does not materially
affect the normal operation of the
biosensor , then the biosensor is
considered to be biocompatible.

24. Detection or Measurement mode (Transducer)—
1. Electrochemical- translate a chemical event to an electrical event by
measuring current passed.
a. Amperometric- most common : movement of electron
produced in a redox reaction.
In 2000 a wearable non-invasive Glucose monitor has been
introduced.
SONY has developed a biofuel cell using sugar as the fuel and
enzymes as catalysts to power a Walkman.
b. Potentiometric -
electrical potential producing phenomenon.

25. 2. Electrical –
a. Conductometric
b. Ion-sensitive –
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.

28. 3. Photochemical –
translate chemical event to a photochemical event,
measure light intensity & wave length.
a. Calorimetric –
measure heat changes of a reaction.
Isothermal ,
Heat conduction , or
Isoperibol .
b. Fluorescence -
property of fluorescence is used.
eg. DNA Microarray
c. Reflectance – use the property of reflection which is detected
by photo detector.

29. 4. Optical –
There is light output during the reaction or a light
absorbance difference between the reactants &
products. It uses property of fluorescence ,
phosphorescence , refraction, and dispersion
spectrometry etc.
a. Fiber optic (optrode/optode)
b. Surface plasmon resonance (SPR)
c. Luminometric
d. Fluorometric

30. 5. Piezoelectric–
These devices are used to detect the specific angle
at which electron waves are emitted when the
substance is exposed to laser light or crystals, such
as quartz ,which vibrate under the influence of an
electric field .
It
translates
a mass change
from a
chemical adsorption event to electrical signal.
The change
in frequency is proportional to
the mass of absorbed material.
a. Acoustic (Surface acoustic wave)
b. Microcantilever
c. Ultrasonic
d. Quartz crystal microbalance (QCM)

31. Nanobiosensors
Nanotechnology will enable us to design
sensors that are much smaller, less
power hungry, and more sensitive than
current micro- or macro sensors.
Examples—
Quantum dots (QD's)
QCM
Microcantilever
Nanoparticles
Nanotubes(CNTs)
Nanowires

32. Ideal Biosensor Characteristics
1. Sensitivity: high ΔS/ Δcanalyte (S = signal)
2. Simple calibration (with standards)
3. Linear Response: ΔS/ Δcanalyte constant over large
concentration range .
4. Accuracy must be there.
5. No hysteresis—signal independent of prior history
of measurements .
6. Response time / Recovery time - less .
7. Selectivity—response only to changes in target
analyte concentration

33. Ideal Biosensor Characteristics –
8. Long-term Stability—not subject to fouling, poisoning, or
oxide formation that interferes with signal;
prolonged stability of biological molecule .
9. Dynamic Response-rapid response to variation in analyte
concentration .
10. Biocompatibility—minimize clotting, platelet interactions,
activation of complement when in
direct contact with bloodstream.
11. Must be cost effective , smaller or portable in size .

34. Applications
1. Medicala. Biosensors are used in both clinical and laboratory use in medical care.
Glucose monitoring in diabetes patients .
Medtronic glucose sensor - implants in major vein of heart.
b. Tumor cells are used as biosensors to monitor the susceptibility of
chemotherapeutic drugs.
c. Routine analytical measurement of folic acid, biotin, vitamin B12 and
pentothenic acid .
d. Micro- and nanoscale biosensors—
Genome mutation detection , cancer detection & clinical diagnosis.
Bacterial-UTI , Human Immunodeficiency Virus (HIV) Detection,
Hepatitis and Anthrax detection.

35. 2. Bio / Pharmaceutical research-
a. Quality assurance
b. Study of biomolecules and their interaction
c. Protein engineering .
d. Drug discovery , evaluation monitor the manufacturing
of biological activity of new compounds (research field).
e. Biosensors are used for measuring concentration of various metal
ions by specific protein concentration or by using genetically
modified organisms.
f. Aptamers are used for the detection of proteins –Thrombin , IgE ,
HIV – tat protein , Lysozyme , Abrin toxin.

36. 2. Bio / Pharmaceutical research-
g. Biosensors are used in monitoring of the glutamate and acetyl
choline , which is the main cause in neurodegenerative diseases.
h. Microbiology: bacterial and viral analysis .
i. Biosensors are used in analysing micro dialysis samples.
j. Biosensors are used in biotechnological process such as to
determine proteins or peptides.
k. Biosensors are also used in determining intracellular proteins and
also plasmids.
l. Detection of cancer biomarkers – CEA , PSA , CA-125 &
Tumour necrosis factor .

37. 3. Industrial / Agricultural –
a. Biosensors used in process control will be able to
measure materials present in the process.
b. Use of biosensors in industry will improve
manufacturing techniques, this will allow for usage of
wider variety of sensing molecules.
c. Biosensors are used in controlling the industrial
processes.
d. Microbial sensor measure Ammonia & Methane.

38. 4. Environmental-
a. Biosensors are used in detecting environmental pollutants
and monitoring of Mines, Industries and toxic gases.
b. Biosensors are used in the BOD measurement during waste
water treatment.
c. Biosensors are used in the detection of poly aromatic
hydrocarbons present in water.
d. Environmental applications e.g. the detection of pesticides
and river water contaminants such as heavy metal ions.
e. Detection and determination of organophosphates .

39. 5. Food industry-
a. Quality assurance in food industries , ex. E. Coli, Salmonella.
b. Food & drink production analysis.
c. Biosensors are used for detection of food freshness marker
determining parameters in wine industry.
d. Determination of drug residues in food, such as antibiotics
and growth promoters, particularly meat and honey.
e. Detection of toxic metabolites such as mycotoxins.

40. 6. Biodefence-
a. Detection of pathogens (SARS in 2003).
b. Remote sensing of airborne bacteria / virus ,
e.g. In counter bio-terrorist activities.
c. Detection system for biological welfare agent
eg. Bacillus anthracis (anthrax) spores.
d. Determining levels of toxic substances before and
after bioremediation.
e. Crime detection.