Transcript test speed rapidity

Application of Electrochemical
Biosensors for Detection of
Food Pathogenic Bacteria
FARAZ KHAN
PHD Scholar
Department of BOTANY
14-ARID-4423
contents
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Introduction
Why to develop biosensors
Phases of general Microbiological methods
Disadvantages of the conventional methods
Summary of the requirements for bacterial biosensors
Microbial Metabolism-Based Biosensors
Antibody-Based Biosensors
Antibody-Based Flow-Injection Biosensors
DNA Biosensors
Future perspective
References
Introduction
“Biosensors are analytical devices incorporating a biological
material (e.g. tissue, microorganisms, organelles, cell receptors,
enzymes, antibodies, nucleic acids etc.), a biologically derived
material or biomimic intimately associated with or integrated
within a physicochemical transducer or transducing microsystem,
which may be optical, electrochemical, thermometric,
piezoelectric, or magnetic.”
Biosensors usually yield a digital electronic signal which is
proportional to the concentration of a specific analyte or group of
analytes. While the signal may in principle be continuous, devices
can be configured to yield single measurements to meet specific
market requirements.”
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Food borne bacteria and use of
biosensors
Escherichia coli, Salmonella typhimurium, Campylobacter jejuni, Legionella
pneumophila, Staphylococcus aureus, Streptococci, etc.) results in numerous
foodborne diseases.
Infectious diseases account for nearly 40% of the total 50 million annual estimated
deaths worldwide.
Unfortunately Pakistan is eighth among the countries, which bears 95% of the
burden of infectious diseases. The main cause of death in over 70% children are
infectious
diseases.
A cursory glance at the leading causes of deaths in children revealed a startling
figure. Large majority of children never reach their fifth birthday.(A.Mehnaz. 2009.
Infectious diseases in children-still leads. J. Med. Assosiation).
Current practices for preventing microbial diseases rely upon careful control of
various kinds of pathogenic bacteria in clinical medicine, food safety and
environmental monitoring
Conventional bacterial identifcation methods usually include a morphological
evaluation of the microorganism as well as tests for the organism's ability to grow in
various media under a variety of conditions.
Phases of general Microbiological
methods
1) Pre-enrichment, to allow growth of all organisms;
2)selective enrichment, to allow growth of the organism under
investigation and to increase bacterial population to a
detectable level;
3)isolation, by using selective agar plates;
4)confirmation, serological and biochemical tests to confirm the
identification of a particular pathogenic organism.
Disadvantages of the conventional
methods
Multistep assay and the time consuming process.
Completion of all phases requires at least 16 h and
can take as long as 48 h.
The detection limit is usually 105-106 cells=ml without
pre-enrichment.
Thus, conventional techniques are not suitable for fast
and direct analysis of bacteria without pre-enrichment.
Summary of the requirements for bacterial
biosensors
Low detection limit :
Ability to detect single bacteria in a reasonably
small sample volume (from 1 to 100 ml).
Species selectivity:
Ability to distinguish individual bacterial species in
the presence of other microorganisms
or cells.
Strain selectivity:
Ability to distinguish an individual bacterial strain
from other strains of the same species.
 Assay time:
• 5 to10 min for a single test.
 Precision:
• 5 to7 %.
 Assay protocol:
• No reagent addition needed.
 Measurement:
• Direct, without pre-enrichment
 Format:
• Highly automated format (``single button
device'').
 Operator:
• No skill required to use the assay.
 Viable cell count:
• Should discriminate between live and dead cells.
 Size:
Compact, portable, hand-held, design for field use
1. Microbial Metabolism-Based
Biosensors
•Microorganisms are able to transduce their metabolic redox
reactions into quantifiable electrical signals by oxidoreductase
reactions using an appropriate mediator, the microbial content of a
sample can be determined by monitoring microbial metabolism.
•Signal parameters such as pH change, oxygen consumption, ion
concentrations, potential difference, current, or resistance, can be
measured by electrochemical transducers.
•The transducer can either detect consumption of oxygen or the
appearance=disappearance of an electrochemical active metabolite.
Identifiable bacterial
species
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E. coli
S. Aureus
Enterococcus serolicida
Gluconobacter industrius
Proteus vulgaris
some drawbacks are:
Biosensors based on the monitoring of microbial
metabolism are related to their poor selectivity
This is because of the possible presence of enzymes
from sources other than the bacteria of interest.
Solution:
 This may be avoided, however, by using antibody
modified sensing elements
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Electrode system consisting of a basal-plane pyrolytic graphite (BPG) electrode
and a porous nitrocellulose membrane Filter to trap bacteria was used for the
detection of bacteria
 Variations in the signals produced by different strains of bacteria.
Antibody based biosensors
• This type of biosensor works by using an antigenantibody reaction to effect a transducer signal
change
• Antibody–based biosensors are a class of
biosensors with high specificity and have the
potential of revolutionizing pathogen detection.
They offer numerous advantages over the
conventional or molecular methodologies, with
the most significant being the option of "on–site"
pathogen detection.
Application of Antibody-Based Biosensors
• A novel liposome-based amperometric biosensor
are used for the detection of haemolytic
microorganisms as
• Haemolytic organisms are able to attack and
rupture the lipid bilayer of liposomes to release
mediator (2,6-dichlorophenolindophenol)
generate a signal, whereas
• a weak or negligible response is produced by
non-haemolytic species.
parameters affecting
these biosensors
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• the binding affinity of the antibody for
antigen
• the nature of the enzyme-marker;
the concentration of conjugate
• and the nature of the transducer.
Bacterial species
• Salmonella
• Staphylococcus aureus
• Escherichia coli
• Listeria monocytogenes
• Listeria welshimeri
3. Antibody-Based Flow-Injection
Biosensors
• This system is based on the selective immunological
separation of Escherichia coli using antibody-coated
magnetic particles and the generation of a signal by
bacterial cells.
• For the immunological step, immunomagnetic
beads were selected as the immunocapture
reagent.
• Electrochemical detection was carried out using
redox mediators, potassium hexacyanoferrate and
2,6-dichlorophenolindoenol.
. One of the problems facing the production of
biosensors for direct detection of bacteria is the
sensitivity of in real samples.
Schematic model of the immunomagnetic separation with
mediated fow injection analysis of viable Escherichia coli O157.
A) Selective capture of E.coli O157 using antibody-derivatized magnetic
particles; B) reaction of bacteria with a mediator; (c) electrochemical
measurement of the reduced mediator using an amperometric method.
• The system has been applied to detect: Bacillus
anthrax spores,
• Escherichia coli O157
• and Salmonella typhimurium.
• The flow-injection immunofltration sensor for
bacteria has four main components:
1. a peristaltic pump,
2. a low pressure sample injection valve (which is
supplied with a fixed volume loop),
3. the amperometric immunosensor assembly,
4. and an electrochemical/data recorder interface.
• The immunosensor consists of a disposable
antibody modified filter membrane resting on top
of the working electrode.
4. DNA Biosensors
• Various kind of electrochemical biosensors
based on identification of the bacterial nucleic acid
have been developed.
• DNA biosensors are analytical devices that
contain immobilized DNA probes that specifically
hybridize to their complementary sequences in a
DNA sample.
• The basic principle of a DNA biosensor is to detect
the molecular recognition provided by the DNA
probe and to transform it into the signal using a
transducer.
• Applications of gene probes are associated
with ultrasensitive determination of
microorganisms,
viruses and trace amounts of special
chemicals in various environments.
• Gene probes are already finding
applications in detection of disease-causing
microorganisms in water supplies, food,
and plant, animal and human tissues.
• Bacterial and viral pathogens responsible for
disease states are detectable because of their
unique nucleic acid sequences.
• Through application of molecular ``probes,'' labeled
DNA sequences that are complementary to unique
portions of bacterial DNA can be detected and
identified.
• The term nucleic acid (gene) probe describes a
segment of nucleic acid which specifically
recognizes and binds to a nucleic acid target.
• Samples containing bacterial nucleic acid are
treated (usually by heating) to cause the double
strands of nucleic acids to separate and thus
become open to hybridization with the nucleic acid
probe.
• Because bacterial nucleic acid may be present in
very small quantities, the probing process is
preceded by a technique called polymerase chain
reaction (PCR), which amplifies the amount of
nucleic acid present.
Areas of applications
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Trace measurements of pollutans
(intercalators, binders of DNA)
Hybridization indicator
(bacteria , virus , genetic inherited diseases)
Biosensing of drugs
• The gene amplification method (the polymerase
chain reaction) enhances the sensitivity of DNA
probes by at least three orders of magnitude .
• This technique uses the heat-stable DNA
polymerase of Thermus aquaticus and allows short
lengths of a double-stranded target DNA (template)
to be copied in vitro thousands or millions of times,
very quickly.
• PCR-gene probe based assay has high potential for
improving monitoring of food born bacteria.
• To date, only methods involving the polymerase
chain reaction (PCR) have been employed to detect
foodborne pathogens.
• Using PCR, bacteria can be detected directly,
without cultivation, by extraction and isolation of
nucleic acids from real samples, followed by
hybridization with specific probes.
• Without any enrichment steps, the PCR method
detects less than 40 cells=gram of a given food
sample.
Examples
• Recently a DNA hybridization electrochemical
biosensor for the detection of DNA fragments of the
waterborne pathogen Cryptosporidium have been
developed.
• Similar hybridization schemes are currently being
developed for other pathogens, such as Escherichia
coli, Giardia and Mycobacterium tuberculosis
Challenges
• The biosensor system must have the
specificity city to distinguish the target
bacteria in a multi-organism matrix, the
adaptability to detect different analytes, the
sensitivity to detect bacteria directly and online without pre-enrichment, and the rapidity
to give real time results. At the same time,
• A biosensor must have relatively simple and
inexpensive configurations.
Future perspective
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in the near future the second generation of
electrochemical biosensors will be fully
automated analytical systems based on
combining multisensor technology with artificial
neural networks ( as in the case of the electronic
nose) or with other analytical and discriminative
mathematical methods.
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The potential of the electronic nose within
the food and drinks industry lies in the speed and
simplicity of the method and in the
nondestructive determination of food quality.
References
1. M.P. Doyle, L.R. Beuchat, T.J. Montville, Food
Microbiology: Fundamentals and Frontiers, ASM
Press, Washington DC, 1997, ch. 3, pp. 127-390.
2. G.W. Beran, H.P. Shoeman, K.F. Anderson, Dairy
Food Environ. Sci. 1991, 11, 189.
3. R.M. Atlas, Critical Rev. Microb. 1998, 24, 157.
4. Malcolm Dando, Biological Warfare in the 21st
Century, Brassey's (UK) London, NY, Macmillan
Pab.Co. 1994.
5. B. Swaminathan, P. Feng, Annu. Rev. Microbiol.
1994, 48, 401.
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