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Microbial biosensors
1
2
Father of the Biosensor
Professor Leland C Clark Jnr
1918–2005
3
Review
4
What is a Biosensor?
in : Biosensor (www.google.com)
International Union of Pure and Applied Chemistry (IUPAC) defines biosensor
as a “device that uses specific biochemical reactions mediated by isolated
enzymes, immunosystems, tissues, organelles or whole cells to detect
chemical compounds usually by electrical, thermal or optical signals”.
INTRODUCTION
• What Is a Biosensor?
☞ Biosensor = bioreceptor + transducer
• The bioreceptor is a biomolecule that recognizes the target analyte
whereas the transducer converts the recognition event into a measurable
signal.
• Enzyme is a Bioreceptor
5
in : Biosensors (www.egtoget.com)
6
Block Diagram of a Biosensor
Sample
(Analyte or
Substrate)
Biorecognition
Element
Transducer
Signal
Processing
Device
Olfactory
Membrane
Olfactory
Nerve Cell
Introduction to Biophotonics – Prasad - John Wiley & Sons © 2003
odor
Brain
7
Block Diagram of a Biosensor
a) Biocatalyst
b) Transducer
c) Amplifier
d) Processor
e) Monitor
8
•Specificity
With biosensors, it is possible to measure specific analytes with great accuracy.
•Speed
Analyte tracers or catalytic products can be directly and instantaneously measured
• Simplicity
Receptor and transducer are integrated into one single sensor& the measurement of
target analytes without using reagents is possible
• Continuous monitoring capability
Biosensors regenerate and reuse the immobilized biological recognition element
in : Biosensors (www.egtoget.com)
9
1. The ability to determine the occurrence of food contamination due to foodborne
pathogens at every stage of food production, processing, and distribution is crucial
to improving the safety of our food supply.
2. There are more than 250 known food borne diseases caused by bacterial and viral
infections.
3. Annually, these foodborne diseases result in an estimated 76 million illnesses,
3,25,000 hospitalizations, 5,000 deaths, and 6 billions dollars in unneeded
expenditure.
4. Bacterial contamination accounts for 91% of total foodborne diseases. Salmonella
sp.,
5. Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Campylobacter
jejuni, Campylobacter coli and Bacillus cereus were found to be main source of
bacterial contaminations in our food supply.
10
11
12
13
14
15
1. A microbial biosensor consists of a transducer in conjunction with
immobilised viable or non-viable microbial cells.
2. Non-viable cells obtained after permeabilisation or whole cells
containing periplasmic enzymes have mostly been used as an
economical substitute for enzymes.
3. Viable cells make use of the respiratory and metabolic functions of the
cell, the analyte to be monitored being either a substrate or an inhibitor
of these processes.
4. Bioluminescence-based microbial biosensors have also been developed
using genetically engineered microorganisms constructed by fusing the
lux gene with an inducible gene promoter for toxicity and
bioavailability testing (7).
16
Advantages
1. Able to metabolise a wide range of chemical compounds
2. Have a great capacity to adapt to adverse conditions and to develop
the ability to degrade new molecules with time.
3. Are also amenable for genetic modifications through mutation or
through recombinant DNA technology and serve as an economical
source of intracellular enzymes.
17
18
19
Fig. Schematic drawing illustrating the wireless nature of the magnetoelastic biosensors and
the basic principle for detecting bacterial cells. The fundamental resonant frequency of the
biosensor is f0 without antigen binding, which shifts (decreases) to fanalyte due to the increased
mass of antigen binding to antibody immobilized on the sensor surface(2).
Salmonell
a
Bacterial binding measurements
1. After the preparation of biosensors, they were exposed to
increasing concentrations (5 * 101 cfu/ml–5 * 108 cfu/ml) of S.
typhimurium spiked in different media (distilled water, fat-free
milk and apple juice) at a flow rate of 100 µl/min.
2. A peristaltic pump (Ismatec Reglo Digital peristaltic pump) was
used to control the flow rate of the media containing the pathogens.
3. The resonance frequency of the sensors was measured using an HP
network analyzer 8751A with S-parameter test set both before and
after the binding of bacterial cells to the immobilized antibody on
the sensor.
4. About of 801 points were recorded over the frequency range with
an 11.31 s sweep time. A standard open circuit calibration was
used to minimize experimental errors in the test set up.
5. A personal computer was used to acquire data at 2 min intervals.
Each data point (Fig. 3) represents the mean values obtained from
five independent sensors, subjected to study under identical
conditions(2).
20
21
1. When the test temperature and humidity are constant, the resonant
frequency change of the magnetoelastic sensor depends only on the mass
change (Δm) on its surface. If the mass increase is small compared to the
mass of the sensor the shift in the resonant frequency is given by:
1. where f is the initial resonance frequency, M is the initial mass, Δm is the
mass change, and Δf is the shift in the resonant frequency of the sensor.
2. Equation shows that the resonance frequency shifts linearly, decreasing
with increasing mass on the sensor surface.
3. Hence binding of the target organism onto the biosensor surface causes a
mass increase with a corresponding decrease in fundamental resonance
frequency(2).
22
23
Sensor surface bacterial densities of 0.105, 0.075 and 0.105
cells/µm2 were observed on the samples which were exposed to S.
typhimurium suspensions in water, fat-free milk, and apple juice
respectively at the highest bacterial concentrations(2).
1. E. coli O157:H7 is most well known to both microbiologists and
general public.
2. This organism was first recognized in 1982, and has the ability to
cause life threatening complications hemorrhagic colitis and hemolytic
uremic syndrome (HUS) in humans(6).
3. Illness due to E. coli O157:H7 infection is often misdiagnosed and
generally needs invasive and expensive medical tests before it is
correctly diagnosed.
4. Primary sources for exposure to E. coli O157:H7 are consumption of
ground beef, sprouts lettuce, salami, unpasteurized milk, and juice
contaminated by pathogens.
5. Since the loss caused by E. coli O157:H7 is enormous in terms of
medical cost and product recall, it is extremely urgent to develop some
rapid and sensitive methods to detect the bacteria in food or water
supplies(6).
24
1. As to the selectivity, DNA is an ideal target for specific detection of
pathogenic bacteria.
2. The DNA probe, which is an oligonucleotide sequence immobilized on
a fixed support and able to hybridize the complementary strand present
in solution, is a powerful molecular tool for monitoring and detecting
specific microorganisms in the environment or in food.
3. It is well known that QCM is a very sensitive mass-measuring sensor,
and the crystal resonance frequency decreases with a mass increase on
the Au electrode surface.
25
26
Nanoparticles are a new class of materials, which has been adopted for
improving the detection limit and sensitivity of the DNA biosensors.
In order to form a more effective and sensitive system, we adopted the gold
nanoparticles in the construction of E. coli O157: H7 DNA biosensor for the
goal of signal amplification, because
(i)colloidal Au has a good biocompatibility;
(ii)gold nanoparticle has a larger surface area, and helps to immobilize more
biofunctional molecules onto the surface of the sensor;
(iii)avidin-conjugated Au nanoparticle could be used as “mass enhancer” to
amplify the frequency change depending on its relatively large mass
compared to DNA target.
27
The whole detection course of this QCM DNA sensor mainly included
two parts: sensor fabrication and detection. During the process of sensor
fabrication, how to immobilize more ssDNA probes was pivotal for the
following bacteria DNA detection, so two important routes for DNA
probes immobilization were introduced to this part, which were:
• Self-assembled monolayers (SAMs).
• Au-thiol binding.
A stepwise decrease of Δf was observed in each fabrication step(6).
28
29
Detection of E.coli in foods
• One of the many applications of surface plasmon
resonance (SPR) technology is the detection of E. coli
O157:H7. Surface plasmon resonance is a quantum
optical-electrical phenomenon based on the fact that
energy carried by photons of light can be transferred
to electrons in a metal. The wavelength of light at
which coupling occurs is characteristic of the particular
metal and the environment of the metal surface
illuminated. This transfer can be observed by
measuring the amount of light reflected by the metal
surface (1, in, 2).
E. coli
The following Fig. shows how a change in the chemical environment
100 nm above the thin metal layer results in a shift in the wavelength of
light, which is absorbed rather than reflected. The most common practical
implementation of SPR is to use a metal-coated optical prism, but other
practical implementations have been demonstrated including metal-coated
diffraction gratings, optical fibres and planar waveguides. The SPR
biosensor has potential for use in rapid, real-time detection and
identification of bacteria, and to study the interaction of organisms with
different antisera or other molecular species (Fratamico et al., 1998). The
lower detection limit of the BIAcore system (a commercial example of an
SPR system) is approximately 10 pg of analyte mm-2.
30
31
E. coli
L. monocytogenes
• L. monocytogenes causes listeriosis, and has been detected in a
variety of environments, including foods. Listeriosis occurs around
the world, with an annual incidence of approximately 7 cases per
million of the normal population. Its incidence tends to be higher in
pregnant women, the elderly, and people with weakened or
suppressed immune systems. The traditional culture – based
technique for the detection of L. monocytogenes requires 5 ~10 days
for complete identification, and is a fairly labor intensive process.
Several alternative detection methods have been developed for the
rapid and sensitive detection of L. monocytogenes , including ELISA,
PCR, DNA microarray, and biosensor methods(4).
32
SPR biosensor technology is one of the most promising detection methods
currently in use for rapid pathogenic identification. It is sensitive to changes in
the thickness or refractive indices of biomaterials at the interface between a
thin gold film and an ambient medium, & is thus capable of characterizing
biomolecular interactions in real time without the need for labeling.
33
As SPR sensors are capable of detecting changes in a refractive index of several
hundred nanometers over the sensor surface, bacterial cell particles specifically
bound to an antibody can induce a large SPR angle shift, as compared with
biomolecules covering an identical surface area(4). The direct detection of microbial
cells is often difficult, owing principally to their large size. In order to assay whole
L.monocytogenes cells with an SPR biosensor, it is first necessary to find a high-
affinity antibody [monoclonal anti- L.monocytogenes mouse antibody] for the cell
and to optimize the detection surface.
In this study, a specific monoclonal antibody against L.monocytogenes was
screened using an SPR biosensor. Monoclonal antibodies were bound to protein L,
after which the L.monocytogenes cells were subjected to an affinity assay. Protein L
was immoblized on a carboxymethyl dextran surface via an amine coupling method,
and utilized repeatedly by regeneration. The monoclonal antibody, ‘A 18’, was
selected and employed for the high sensitivity detection of L.monocytogenes.
specific antibodies were screened via direct binding to cells using an SPR biosensor.
It was determined that the screened antibody evidenced a high degree of sensitivity
for the direct detection of L.monocytogenes.
34
• In a study that was done in year 2007 by Joung Hyou- Arm et al., they
selected a specific monoclonal antibody (mouse IgG1) against L.
monocytogenes , and also attempted to detect it directly. The direct
detection of microbial cells is often difficult, owing to principally large
size. In order to assay whole L. monocytogenes cells with a SPR
biosensor, it is first necessary to find a high- affinity antibody for the cell
& to optimize the detection surface (4).
35
References
1. Maria N. Velasco-Garcia; Toby Mottram (2003). Biosensor Technology
addressing Agricultural Problems. Biosystems Engineering , 84 (1), 1–12
2. Guntupalli, R., Lakshmanan, Ramji S., Johnson, Michael L., Hu, J., Huang,
Tung-Shi., Barbaree, James M., Vodyanoy, Vitaly J., Chin, Bryan A. (2007).
Magnetoelastic biosensor for the detection of Salmonella typhimurium in food
products. Sens. & Instrumen. Food Qual. 1:3–10 DOI 10.1007/s11694-006-9003-
8
3. SKLADAL, P., SYMERSKA, Y., POHANKA, M., SAFAR, B., MACELA, A.
(2005). ELECTROCHEMICAL IMMUNOSENSOR FOR DETECTION OF
FRANCISELLA TULARENSIS. Detection Technologies, Implementation
Strategies and Commercial Opportunities, 221–232.
4. Joung, H., et al. (2007). Screening of a Specific Monoclonal Antibody against
and Detection of Listeria monocytogenes Whole Cells Using a Surface Plasmon
Resonance Biosensor. Biotechnology and Bioprocess Engineering, 12 : 80-85.
5. Nayak, M., Kotian, A., Marathe, S., Chakravortty, D. (2009). Detection of
microorganisms using biosensors—A smarter way towards detection techniques.
Biosensors and Bioelectronics 25, 661–667.
6. LiJiang, W., QingShan, W., ChunSheng, W., ZhaoYing, H., Jian, J. & Ping, W.
(2008). The Escherichia coli O157:H7 DNA detection on a gold nanoparticle-
enhanced piezoelectric biosensor. Chinese Science Bulletin, vol. 53 | no. 8 | 1175-
1184.
7. D’Souza, S.F.(2001). Microbial biosensors. Biosensors & Bioelectronics 16,
337–353.
36
Any Questions???
37

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Environmental Biotechnology Topic:- Microbial Biosensor

  • 2. 2 Father of the Biosensor Professor Leland C Clark Jnr 1918–2005
  • 4. 4 What is a Biosensor? in : Biosensor (www.google.com) International Union of Pure and Applied Chemistry (IUPAC) defines biosensor as a “device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals”.
  • 5. INTRODUCTION • What Is a Biosensor? ☞ Biosensor = bioreceptor + transducer • The bioreceptor is a biomolecule that recognizes the target analyte whereas the transducer converts the recognition event into a measurable signal. • Enzyme is a Bioreceptor 5 in : Biosensors (www.egtoget.com)
  • 6. 6 Block Diagram of a Biosensor Sample (Analyte or Substrate) Biorecognition Element Transducer Signal Processing Device Olfactory Membrane Olfactory Nerve Cell Introduction to Biophotonics – Prasad - John Wiley & Sons © 2003 odor Brain
  • 7. 7 Block Diagram of a Biosensor a) Biocatalyst b) Transducer c) Amplifier d) Processor e) Monitor
  • 8. 8 •Specificity With biosensors, it is possible to measure specific analytes with great accuracy. •Speed Analyte tracers or catalytic products can be directly and instantaneously measured • Simplicity Receptor and transducer are integrated into one single sensor& the measurement of target analytes without using reagents is possible • Continuous monitoring capability Biosensors regenerate and reuse the immobilized biological recognition element in : Biosensors (www.egtoget.com)
  • 9. 9 1. The ability to determine the occurrence of food contamination due to foodborne pathogens at every stage of food production, processing, and distribution is crucial to improving the safety of our food supply. 2. There are more than 250 known food borne diseases caused by bacterial and viral infections. 3. Annually, these foodborne diseases result in an estimated 76 million illnesses, 3,25,000 hospitalizations, 5,000 deaths, and 6 billions dollars in unneeded expenditure. 4. Bacterial contamination accounts for 91% of total foodborne diseases. Salmonella sp., 5. Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Campylobacter jejuni, Campylobacter coli and Bacillus cereus were found to be main source of bacterial contaminations in our food supply.
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  • 16. 1. A microbial biosensor consists of a transducer in conjunction with immobilised viable or non-viable microbial cells. 2. Non-viable cells obtained after permeabilisation or whole cells containing periplasmic enzymes have mostly been used as an economical substitute for enzymes. 3. Viable cells make use of the respiratory and metabolic functions of the cell, the analyte to be monitored being either a substrate or an inhibitor of these processes. 4. Bioluminescence-based microbial biosensors have also been developed using genetically engineered microorganisms constructed by fusing the lux gene with an inducible gene promoter for toxicity and bioavailability testing (7). 16
  • 17. Advantages 1. Able to metabolise a wide range of chemical compounds 2. Have a great capacity to adapt to adverse conditions and to develop the ability to degrade new molecules with time. 3. Are also amenable for genetic modifications through mutation or through recombinant DNA technology and serve as an economical source of intracellular enzymes. 17
  • 18. 18
  • 19. 19 Fig. Schematic drawing illustrating the wireless nature of the magnetoelastic biosensors and the basic principle for detecting bacterial cells. The fundamental resonant frequency of the biosensor is f0 without antigen binding, which shifts (decreases) to fanalyte due to the increased mass of antigen binding to antibody immobilized on the sensor surface(2). Salmonell a
  • 20. Bacterial binding measurements 1. After the preparation of biosensors, they were exposed to increasing concentrations (5 * 101 cfu/ml–5 * 108 cfu/ml) of S. typhimurium spiked in different media (distilled water, fat-free milk and apple juice) at a flow rate of 100 µl/min. 2. A peristaltic pump (Ismatec Reglo Digital peristaltic pump) was used to control the flow rate of the media containing the pathogens. 3. The resonance frequency of the sensors was measured using an HP network analyzer 8751A with S-parameter test set both before and after the binding of bacterial cells to the immobilized antibody on the sensor. 4. About of 801 points were recorded over the frequency range with an 11.31 s sweep time. A standard open circuit calibration was used to minimize experimental errors in the test set up. 5. A personal computer was used to acquire data at 2 min intervals. Each data point (Fig. 3) represents the mean values obtained from five independent sensors, subjected to study under identical conditions(2). 20
  • 21. 21
  • 22. 1. When the test temperature and humidity are constant, the resonant frequency change of the magnetoelastic sensor depends only on the mass change (Δm) on its surface. If the mass increase is small compared to the mass of the sensor the shift in the resonant frequency is given by: 1. where f is the initial resonance frequency, M is the initial mass, Δm is the mass change, and Δf is the shift in the resonant frequency of the sensor. 2. Equation shows that the resonance frequency shifts linearly, decreasing with increasing mass on the sensor surface. 3. Hence binding of the target organism onto the biosensor surface causes a mass increase with a corresponding decrease in fundamental resonance frequency(2). 22
  • 23. 23 Sensor surface bacterial densities of 0.105, 0.075 and 0.105 cells/µm2 were observed on the samples which were exposed to S. typhimurium suspensions in water, fat-free milk, and apple juice respectively at the highest bacterial concentrations(2).
  • 24. 1. E. coli O157:H7 is most well known to both microbiologists and general public. 2. This organism was first recognized in 1982, and has the ability to cause life threatening complications hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans(6). 3. Illness due to E. coli O157:H7 infection is often misdiagnosed and generally needs invasive and expensive medical tests before it is correctly diagnosed. 4. Primary sources for exposure to E. coli O157:H7 are consumption of ground beef, sprouts lettuce, salami, unpasteurized milk, and juice contaminated by pathogens. 5. Since the loss caused by E. coli O157:H7 is enormous in terms of medical cost and product recall, it is extremely urgent to develop some rapid and sensitive methods to detect the bacteria in food or water supplies(6). 24
  • 25. 1. As to the selectivity, DNA is an ideal target for specific detection of pathogenic bacteria. 2. The DNA probe, which is an oligonucleotide sequence immobilized on a fixed support and able to hybridize the complementary strand present in solution, is a powerful molecular tool for monitoring and detecting specific microorganisms in the environment or in food. 3. It is well known that QCM is a very sensitive mass-measuring sensor, and the crystal resonance frequency decreases with a mass increase on the Au electrode surface. 25
  • 26. 26 Nanoparticles are a new class of materials, which has been adopted for improving the detection limit and sensitivity of the DNA biosensors. In order to form a more effective and sensitive system, we adopted the gold nanoparticles in the construction of E. coli O157: H7 DNA biosensor for the goal of signal amplification, because (i)colloidal Au has a good biocompatibility; (ii)gold nanoparticle has a larger surface area, and helps to immobilize more biofunctional molecules onto the surface of the sensor; (iii)avidin-conjugated Au nanoparticle could be used as “mass enhancer” to amplify the frequency change depending on its relatively large mass compared to DNA target.
  • 27. 27 The whole detection course of this QCM DNA sensor mainly included two parts: sensor fabrication and detection. During the process of sensor fabrication, how to immobilize more ssDNA probes was pivotal for the following bacteria DNA detection, so two important routes for DNA probes immobilization were introduced to this part, which were: • Self-assembled monolayers (SAMs). • Au-thiol binding. A stepwise decrease of Δf was observed in each fabrication step(6).
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  • 29. 29 Detection of E.coli in foods • One of the many applications of surface plasmon resonance (SPR) technology is the detection of E. coli O157:H7. Surface plasmon resonance is a quantum optical-electrical phenomenon based on the fact that energy carried by photons of light can be transferred to electrons in a metal. The wavelength of light at which coupling occurs is characteristic of the particular metal and the environment of the metal surface illuminated. This transfer can be observed by measuring the amount of light reflected by the metal surface (1, in, 2). E. coli
  • 30. The following Fig. shows how a change in the chemical environment 100 nm above the thin metal layer results in a shift in the wavelength of light, which is absorbed rather than reflected. The most common practical implementation of SPR is to use a metal-coated optical prism, but other practical implementations have been demonstrated including metal-coated diffraction gratings, optical fibres and planar waveguides. The SPR biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with different antisera or other molecular species (Fratamico et al., 1998). The lower detection limit of the BIAcore system (a commercial example of an SPR system) is approximately 10 pg of analyte mm-2. 30
  • 32. L. monocytogenes • L. monocytogenes causes listeriosis, and has been detected in a variety of environments, including foods. Listeriosis occurs around the world, with an annual incidence of approximately 7 cases per million of the normal population. Its incidence tends to be higher in pregnant women, the elderly, and people with weakened or suppressed immune systems. The traditional culture – based technique for the detection of L. monocytogenes requires 5 ~10 days for complete identification, and is a fairly labor intensive process. Several alternative detection methods have been developed for the rapid and sensitive detection of L. monocytogenes , including ELISA, PCR, DNA microarray, and biosensor methods(4). 32
  • 33. SPR biosensor technology is one of the most promising detection methods currently in use for rapid pathogenic identification. It is sensitive to changes in the thickness or refractive indices of biomaterials at the interface between a thin gold film and an ambient medium, & is thus capable of characterizing biomolecular interactions in real time without the need for labeling. 33
  • 34. As SPR sensors are capable of detecting changes in a refractive index of several hundred nanometers over the sensor surface, bacterial cell particles specifically bound to an antibody can induce a large SPR angle shift, as compared with biomolecules covering an identical surface area(4). The direct detection of microbial cells is often difficult, owing principally to their large size. In order to assay whole L.monocytogenes cells with an SPR biosensor, it is first necessary to find a high- affinity antibody [monoclonal anti- L.monocytogenes mouse antibody] for the cell and to optimize the detection surface. In this study, a specific monoclonal antibody against L.monocytogenes was screened using an SPR biosensor. Monoclonal antibodies were bound to protein L, after which the L.monocytogenes cells were subjected to an affinity assay. Protein L was immoblized on a carboxymethyl dextran surface via an amine coupling method, and utilized repeatedly by regeneration. The monoclonal antibody, ‘A 18’, was selected and employed for the high sensitivity detection of L.monocytogenes. specific antibodies were screened via direct binding to cells using an SPR biosensor. It was determined that the screened antibody evidenced a high degree of sensitivity for the direct detection of L.monocytogenes. 34
  • 35. • In a study that was done in year 2007 by Joung Hyou- Arm et al., they selected a specific monoclonal antibody (mouse IgG1) against L. monocytogenes , and also attempted to detect it directly. The direct detection of microbial cells is often difficult, owing to principally large size. In order to assay whole L. monocytogenes cells with a SPR biosensor, it is first necessary to find a high- affinity antibody for the cell & to optimize the detection surface (4). 35
  • 36. References 1. Maria N. Velasco-Garcia; Toby Mottram (2003). Biosensor Technology addressing Agricultural Problems. Biosystems Engineering , 84 (1), 1–12 2. Guntupalli, R., Lakshmanan, Ramji S., Johnson, Michael L., Hu, J., Huang, Tung-Shi., Barbaree, James M., Vodyanoy, Vitaly J., Chin, Bryan A. (2007). Magnetoelastic biosensor for the detection of Salmonella typhimurium in food products. Sens. & Instrumen. Food Qual. 1:3–10 DOI 10.1007/s11694-006-9003- 8 3. SKLADAL, P., SYMERSKA, Y., POHANKA, M., SAFAR, B., MACELA, A. (2005). ELECTROCHEMICAL IMMUNOSENSOR FOR DETECTION OF FRANCISELLA TULARENSIS. Detection Technologies, Implementation Strategies and Commercial Opportunities, 221–232. 4. Joung, H., et al. (2007). Screening of a Specific Monoclonal Antibody against and Detection of Listeria monocytogenes Whole Cells Using a Surface Plasmon Resonance Biosensor. Biotechnology and Bioprocess Engineering, 12 : 80-85. 5. Nayak, M., Kotian, A., Marathe, S., Chakravortty, D. (2009). Detection of microorganisms using biosensors—A smarter way towards detection techniques. Biosensors and Bioelectronics 25, 661–667. 6. LiJiang, W., QingShan, W., ChunSheng, W., ZhaoYing, H., Jian, J. & Ping, W. (2008). The Escherichia coli O157:H7 DNA detection on a gold nanoparticle- enhanced piezoelectric biosensor. Chinese Science Bulletin, vol. 53 | no. 8 | 1175- 1184. 7. D’Souza, S.F.(2001). Microbial biosensors. Biosensors & Bioelectronics 16, 337–353. 36