Biosensors: Amperometric:Blood-glucose biosensor:Amperometric:Potentiometric Biosensors:Conductometric Biosensors:Optical Biosensors:Therapeutic Applications of Immobilized Enzymes:

1. Biosensors:
By
Dr. Harinatha Reddy A
ASSISTANT PROFESSOR
Department of Biotechnology
biohari14@gmail.com
Bangalore

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4. Introduction:
 Biosensor: is an analytical device containing an immobilized
biological material (Enzyme, antibody, nucleic acid, hormones or
whole cell).
 Which can be interact with an analyte produce physical, chemical
or electrical signals that can be measured.

5.  Biosensors basically involve the quantitative analysis of various
substances by converting their biological actions into measurable
signals.
 A great majority of biosensors have immobilized enzymes.

6. A biosensor has two main components…………
1. Biological component—Immobilized enzyme, antigen or antibody
or cell etc.
2. Physical component —Transducer.

8.  The biological component recognises and interacts with the analyte
to produce a physical change (a signal) that can be detected, by the
transducer.
 In practice, the biological material is appropriately immobilized on
to the transducer.
 A transducer is a device that converts energy from one form to
another. Usually a transducer converts a signal in one form of
energy to a signal in another.

9. Types of Biosensors:
 Amperometric
 Potentiometric
 Conductometric,
 Optical Biosensors

10. Amperometric:

11. Blood-glucose biosensor:
(Glucose oxidase)
Glucose+ O2-------- ------------------- Gluconolactone+ H2O2
Oxygen
electrode

12. Blood-glucose biosensor:
Glucose+ O2---------------------------Gluconolactone+ H2O2
Oxygen
electrode

13. Amperometric:
 These biosensors are based on the movement of electrons (i.e.
determination of electric current) as a result of enzyme-catalysed
redox reactions.
 Normally, a constant voltage passes between the electrodes which
can be determined.
 In an enzymatic reaction that occurs, the substrate or product can
transfer an electron with the electrode surface to be oxidised or
reduced..

14.  This results in an altered current flow that can be measured. The
magnitude (size) of the current is proportional to the substrate
concentration.
 Blood-glucose biosensor is example for Amperometric
biosensors: used for Determination of glucose.

15. Blood-glucose biosensor:
• It is a good example of amperometric biosensors, widely used
throughout the world by diabetic patients.
• Blood- glucose biosensor looks like a watch pen and has a single
use disposable electrode (consisting of a Ag/AgCI reference
electrode and a carbon working electrode) with glucose oxidase
enzyme.
• Reduction of oxygen at the surface of the cathode causes the
oxygen concentration there to be effectively zero. It is clear that a
small, but significant, proportion of the oxygen present in the bulk
is consumed by this process; the oxygen electrode measuring the
rate of a process which is far from equilibrium.

16. Potentiometric Biosensors:

17. Potentiometric Biosensors:
Substrate Product
Ion- selective electrodes used
To determine H+ ion con.

18. Potentiometric Biosensors:
 In these biosensors,
changes in ionic
concentrations (H+) are
determined by use of ion-
selective electrodes.
 since many enzymatic
reactions involve the
release or absorption of
hydrogen ions.
 pH electrode is the most
commonly used ion-
selective electrode,
Substrate Product

19. Potentiometric Biosensors:
 In these biosensors, changes in ionic concentrations (H+) are
determined by use of ion- selective electrodes.
 pH electrode is the most commonly used ion-selective electrode,
 since many enzymatic reactions involve the release or absorption of
hydrogen ions.

20. There are different types of ion-selective electrodes which are of use
in biosensors:
 Glass electrodes for H+ ions: (e.g. normal pH electrodes):
penicillinase
penicillin----------------------- penicilloic acid + H+
lipase
neutral lipids + H2O.................. glycerol + fatty acids + H+

21.  Glass electrodes for CO2, NH3 or H2S.
asparaginase `
L-asparagine + H2O-------------------- L-aspartate + NH4
+ or NH3
 The iodide electrode is useful for the determination of I- in the
peroxidase reaction.
peroxidase
H2O2 + 2H+ + 2I- ------------------- I2 + 2H2O

22. There are three types of ion-selective electrodes which are of use in
biosensors:
 Glass electrodes for cations (+) (e.g. normal pH electrodes): It
contain gas permeable membrane selective for H+ Ions. The
diffusion of H+ ions through the membrane causes changes in pH
of sensing solution. It can be measure by electrodes.
 Glass pH electrodes coated with a gas-permeable membrane
selective for CO2, NH3 or H2S. The diffusion of the gas through
this membrane causes a change in pH of a sensing solution between
the membrane and the electrode which is then determined.
 The iodide electrode is useful for the determination of I- in the
peroxidase reaction (Table 6.2c) and also responds to cyanide ions.

23. Conductometric Biosensors:

24. Conductometric Biosensors:
Urea biosensor
OH-

25. Conductometric Biosensors:
 These ionic species alter the electrical conductivity which can be
measured.
 There are several reactions in the biological systems that bring about
changes in the ionic species.
 A good example of conduct metric biosensor is the urea biosensor
utilizing immobilized urease.
 The above reaction is associated with drastic alteration in ionic
concentration which can be used for monitoring urea concentration.
 In fact, urea biosensors are very successfully used during dialysis and
renal surgery.

26. Optical Biosensors:

27. Fibre optic lactate biosensor:
Lactate dehydrogenase (LDH) has been directly immobilized
onto an optical fiber probe..

28. Optical Biosensors for Blood Glucose:
Glucose oxidase + Horse radish peroxidase+ Chromogen
Portable reflectance meter.
Glucose oxidase
Glucose --- -------------- ------Gluconic acid + H2O2
Peroxidase
Chromogen+ 2H202 -------------------- Colour dye + 2H20+ O2

29. Luminescent biosensors to detect urinary infections:
Leuciferin + ATP+O2
Oxyluciferin+ CO2+ AMP+ LIGHT
Luciferase
Leuciferin+ Luciferase

30. Optical Biosensors:
 Optical biosensors are the devices that utilize the principle of optical
measurements (absorbance, fluorescence, chemiluminescence etc.).
 They employ the use of fibre optics and optoelectronic transducers.
 Some of the important optical biosensors are briefly described here
under.

31. Example: Fibre optic lactate biosensor:
 In Fibre optic lactate biosensors Lactate dehydrogenase (LDH) has
been directly immobilized onto an optical fiber probe surface through
covalent binding.
 The amount of fluorescence generated by the dyed film is dependent on
the O2.
 This is because O2 has a quenching (reducing) effect on the fluorescence.
 As the concentration of lactate in the reaction mixture increases, O2 is
utilized, and consequently there is a proportionate decrease in the
quenching effect.
 The result is that there is an increase in the fluorescent output which can
be measured.

32. Optical Biosensors for Blood Glucose:
 Estimation of blood glucose is very important for monitoring of
diabetes.
 A simple technique involving paper strips impregnated with
reagents is used for this purpose.
 The strips contain glucose oxidase, horse radish peroxidase and a
chromogen (e.g. toluidine).
 The intensity of the colour of the dye can be measured by using a
portable reflectance meter.
 Colorimetric test strips of cellulose coated with appropriate enzymes
and reagents are in use for the estimation of several blood and urine
parameters.

33. Luminescent biosensors to detect urinary infections:
 The microorganisms in the urine, causing urinary tract infections,
can be detected by employing luminescent biosensors. For this
purpose, the immobilized (or even free) enzyme namely luciferase is
used. The microorganisms, on lysis release ATP which can be
detected by the following reaction. The quantity of light output
can be measured by electronic devices.

34. Therapeutic Applications of Immobilized Enzymes:

35.  Immobilized streptokinase and urokinase can be used for the
treatment of thromboses.
 Immobilized Phenylalanine hydroxylase to treat phenylketonuria.
 Immobilized Lysosomal α 1, 4-glucosidase to correct type II
glycogen storage disease.

36. Immobilized enzymes in drug delivery:

37.  Urea-urease modulated system:
Poly venyl methyl ether is a
biodegradable polymer
+ OH-

38.  Urea-urease modulated system:

39. Glucose oxidase-glucose modulated system:
INSULIN
Glucose
oxidase

40. Glucose oxidase-glucose modulated system:
 For insulin delivery to the human body, a bio-degradable polymeric
system containing insulin has been developed.
 The delivery of insulin can be modulated by immobilized glucose
oxidase enzyme.
 As glucose oxidase acts on glucose, gluconic acid is produced
which lowers the pH.
 The low pH in turn, causes the release of insulin from the bio-
degradable polymeric system.

41.  How are immobilized enzymes useful as biosensors?
 What is biosensor? Explain conductometric and optical biosensors
with diagrams and their applications?
 Explain Amperometric and Potentiometric and their applications?
 Explain role of immobilised enzymes in drug delivery.

42. THANK YOU: