Electrochemical Microbial

Biosensor
Presented by-
Ruchi
M.Sc (F)
 Biosensor
• Biosensor basically consists of transducer in
conjunction with a biologically active molecule
that converts a biochemical signal into a
quantifiable electric response.
• Some of the major attributes of a good biosensing
system are its repeatability, reproducibility,
specificity, sensitivity, reliability, portability, ability
to function even in opaque solution, good
response time and simplicity in operation.
 Microbial biosensor
• A microbial biosensor consists of a transducer in
conjunction with immobilized viable or non-viable
microbial cells.
• Viable cells make use of respiratory and metabolic
functions of the cell, analyte can be a substrate or
inhibitor of these processes. Ex. BOD biosensor.
• Non viable cells obtained after permeabilization
or whole cells containing periplasmic enzymes
have mostly been used as an economical
substitute for enzymes.
 ELECTROCHEMICAL MICROBIAL
BIOSENSOR
• It measures electrical signal, which is generated during
the biochemical interaction between the biologically
active part and substrate.
• It generally consists of a working electrode, a
transducer layer for detection (microorganism) and
recording equipment.
• Signal from the transducer, produced by the
electrochemical reaction is recorded and correlated
with the concentration and correlation with the
concentration and composition of the compound
present and displayed as an electrical expression.
Types of electrochemical biosensor
• Amperometric biosensor- involves measuring
the current generated by electrochemical
oxidation or reduction of electroactive species
at a constant applied potential.
• Potentiometric biosensor- measures the
electric potential difference between a
working and reference electrode.
 Types of Electrochemical biosensor
• Conductometric biosensor- measures changes
in the conductance of the system due to
presence of the analyte. Ex - urea biosensors,
with immobilized Brevibacterium
ammoniagenes very successfully used during
dialysis and renal surgery.
• Voltametric biosensor- they record and
correlate each electric signal (electric current
and potential difference) with a
corresponding sample.biosensor using
Circinella sp. for detection of Copper ions.
Amperometric glucose biosensor
WORKING OF GLUCOSE BIOSENSOR
 Potentiometric Biosensor
• Biosensor constructed by modifying a pH
electrode with an immobilizing layer of
Escherichia coli cells expressing
organophosphorous hydrolase (OPH) on the cell
surface.
• OPH catalyzes the hydrolysis of
organophosphorous pesticides
to release two protons,
the concentration of which is
proportional to the amount
of hydrolyzed substrate.
• Potentiometric
biosensors have ion
selective electrodes
that detect a change
in voltage
depending on the
analyte
concentration.
 Operating methods
• The batch method- biosensors are usually
capable of determining a compound directly
in a sample medium.
• Flow injection analysis- it is used when the
analyte is in a flowing liquid. The sample may
be directly injected into a continuous flow of
carrier liquid without air segmentation.
 Applications
• Short chain fatty acids cause rancidity of milk. Arthrobacter
nicotianae has been shown to possess enzymes of the beta
oxidation pathway with a high specificity towards short chain
fatty acids.
• Sachharomyces uvarum biosensor uses it’s respiratory activity
for detection of vitamin B-6.
• In pharmaceuticals and medicine, they are used for the
estimation of steroids such as cholestrol (Nocardia
erythropolis), androstendione
(N. erythropolis), testosterone (N. erythropolis); antibiotics
such as nystatin (S. cereisiae) and other compounds like
gonadotropin releasing hormone. (B. subtilis).
 Advantages
• Ubiquitous and able to metabolize a wide range of
chemical compounds.
• Microorganisms have a great capacity to adapt to
adverse conditions and to develop the ability to
degrade new molecules with time.
• Avoids lengthy and expensive operation of
enzyme purification.
• Preserves the enzymes in its natural environment
and protects it from inactivation by external
toxicants such as metals.
• Amenable for genetic modification.
 Advantages
• Regeneration of cofactor is not required.
• These biosensors are stable, longer half lives.
• Less sensitive to variation in pH and
temperature.
 Disadvantages
• Slow response due to diffusion barriers.
• low specificity.
• Low sensitivity.
• Instrumental limitation.
*selection of appropriate culture.
 Solutions
➢ Use of permeabilized cells:
• -the cells can be permeabilized using physical (freezing or
thawing), chemical (toluene, chloroform) etc. such
treatment create minute pores by removing some of the
lipids from the cell membrane. It removes diffusion
barriers.
-permeabilization also remove small molecular weight
cofactors thus minimizing side reactions.
• Surface expressed enzymes: periplasmic invertase and
catalase in yeast.
• Inactivating other enzymes in case of non viable cells used.
• The blockage of undesired transport mechanism.
• Hybrid sensor: Gluconobacter oxydans
containing glucose oxidase has been used in
conjunction with Sachharomyces cerevisiae
cells containing periplasmic invertase in the
fabrication of sucrose biosensor.
 Institutions
• Nuclear Agricultural and Biological Division,
Bhabha atomic Research Centre, Trombay,
Mumbai.
• Department of Chemical and Environmental
Engineering, University of California.
• Department of Chemical, Materials and
Biomolecular Engineering, University of
Connecticut.
• Biodesign Institute, Center for Bioelectronics and
Biosensors, Departments of Chemical Engineering
and Chemistry and Biochemistry,
 References
• Miertus S, Kartlik J, Pizzariello A, Stedansky M, Svitel J,
Svorc J. Amperometric biosensors based on solid binding
matrices applied in food quality monitoring. Biosens
Bioelectron 1998; 13: 911-924.
• Rotariu, L.; Bala, C. Microbial biosensor for ethanol
determinationin alcoholic beverages. Bucharest University
Annals—Chemistry in press, 2003, I–II, 69–76.
• D’Souza, S.F. Microbial biosensors. Biosens. Bioelectron.
2001, 16,337–353.
• Methods in Biofechnology, Vol. 6: Enzyme and Microbial
Biosensors: Techniques and Protocols Edited by: A.
Mulchandani and K. R. Rogers 0
 Questions
1.What is the principle of electrochemical
biosensor?
2.What is the role of oxygen in glucose biosensor?
3.Oxidation of hydrogen peroxide occurs at
.
4.What are the disadvantages of microbial
biosensor?
5.Give the strategies to resolve the problems
associated with microbial biosensor?
6.Name one mediator used in second generation
glucose biosensor.
 Answers
1. Signal from the transducer, produced by the
eletrochemical reaction is measured and
correlated with the concentration of the
compound.
2. 2. oxygen acts as an electron acceptor for
reoxidation of FADH2.
3. anode.
4. (a) slow process, (b) low selectivity, (c) low
sensitivity.
 Answers
5. use of permeabilized cells, use of cells with
surface expressed enzymes, inactivation of
other enzymes in the cells.
6. ferrocene.